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Modern Spectroscopy Lab

Fluorescence Spectroscopy

Fluorescence spectroscopy is a methodology that uses a beam of electromagnetic radiation to excite the electrons in certain molecules, and such excitation causes the molecules to emit light. The excitation wavelength is typically (not always) part of the ultraviolet spectrum, while the emitted light is most often (not always) in the visible spectrum. Thus, by definition fluorescence is the visible light emitted by certain substances as a result of incident radiation of a shorter wavelength.

In order for a molecule to emit a photon of a given energy through the mechanism of fluorescence it must first exist in a given state defined by quantum physics.  Most molecules (at room temperature) will exist in their equilibrium ground state as a so-called singlet (S0), with a full complement of paired electrons.  Incident light of sufficient energy might raise the system energy to a higher electronic singlet; S1 or S2 for example.  While an electron is excited to a higher energy orbital in S1 and S2, the spin of the now unpaired electron remains antiparallel, in contrast to transitions in an excited triplet (see below) where we find the unpaired electron in the higher energy triplet orbital retains the parallel configuration. 

Of note there are typically many vibrational (V1-N), and rotational levels that might be occupied above a particular electronic singlet.  (The rotational levels will be ignored, but of note the entire family of possible vibrational and rotational energies gives rise to the smooth character of the recorded spectra.)  Thus, an electron might be promoted to any vibrational state whose energy lies above the respective electronic singlet SN(the so-called Franck-Condon excited state; see Ref. 1 and 2, and the figure below).  If the electron is promoted to a vibrational level above S1 (or SN) a radiation-less process called vibrational relaxation (VR) occurs whereby the electron loses thermal energy to its environment and in rapid time will attain S1, or SN.  If the electron is promoted to S2 or higher (SN) VR may occur through one or more excited singlets in its relaxation to S1, or another radiation-less process known as internal conversion (IC) (see below) might contribute to its relaxation to S1.

Fluorescence may now occur when photons decay from the lowest vibrational state of the first excited singlet, S1(the equilibrium excited state) to the Franck-Condonground state, S0VN.  Fluorescent decay does not occur directly from a higher order singlet to a ground state; for example, from S2 to S0.  A photon is emitted only when the electrons are in the lowest (vibrational) energy configuration of the first excited singlet (S1). However, the emitted photon might occupy any of the vibrational energy orbitals that exist between S1 and S0 (the Franck-Condon ground state). 


The Jablonski Diagram shown below is a recapitulation of the above information.  The absorption of a photon of sufficient energy might increase the energy of S0 to any one of many possible vibrational/rotational energies associated with a particular excited state singlet (SNV1-VN).  If a given photon promotes an electron to a higher vibrational level of a given singlet, for example S1V4 or S2V3, or more generally to (SNVN), emission of fluorescent light will occur only after VR occurs through each of the vibrational energy states, to (S1V0).  Thus, emission will occur only from the lowest vibrational state (V0) of the first excited singlet; (S1V0).  However, the emitted photon may target any allowed ground state vibrational configuration; (S0 –V0-N), the Franck-Condon ground state. For example, a photon may arrive at the third vibrational energy in the ground state (e.g., S0V3) and still fluoresce. 

In the event that the excited electron occupies a vibrational orbital above S2 (or SN), VR occurs to S2-N-V0 and a process known as IC usually occurs prior to further VR to S1V0.  In IC electrons may pass from the lowest vibrational level of the higher electronic state to a higher, degenerate (unoccupied) orbital of a lower excited state that has the same energy as the orbital from which it came.  Further VR then occurs until the S1V0 condition is achieved.  If relaxation must occur from S3 or higher singlets several ICs may be required.  This is very common for most fluorescent molecules.  (In the figure below the process is demonstrated for S2V3.)  Thus, excited state electrons undergo rapid thermal energy loss to their molecular environment through serial VRs, often requiring one or more ICs, and a photon is emitted from the lowest-lying excited singlet vibrational state.

In the diagram depicted below energy state transitions are characterized either by strait lines or wavy lines.  The strait lines represent transitions associated with the absorption or emission of a photon.  The wavy lines represent processes that occur without the emission of light (radiation-less).  These include the VRs and ICs already discussed. It is noted that depending on the fluorochrome and the excitation energy the excited state singlet might be an S1, an S2 or higher.  Therefore, both types of relaxation are typically required to achieve the lowest vibrational energy of S1 (S1V0), which is required for the emission of a photon. 

Other radiation-less mechanisms of energy loss occur, such as the intersystem crossing (ISC),which is an isoenergetic transition between two electronics states having different electron spin multiplicities.  When a singlet passes to a triplet state the spin of the excited electron is reversed.  However, the direct transition from a singlet in the ground state (S0) to a triplet orbital is forbidden by the rules of quantum mechanics. On the other hand, it is possible for electron transfer to occur from the lowest excited singlet state (S1) to one of the higher vibrational levels in a triplet configuration, providing energies are equal. This is typically a slow process, much slower (10⁻² – 10⁻⁴ sec) than the process of fluorescence, which requires only a nanosecond (these are approximate relative times).   But it should be noted that the least energetic triplet configuration has less energy than the equilibrium singlet ground state. Thus, in those conditions where intersystem crossing occurs from an excited singlet whose energy approximates the orbital energy of a higher vibrational state triplet, vibrational relaxation will occur to T1, and it is possible for yet another intersystem crossing to occur back to S0, the equilibrium singlet ground energy. While this adds additional time, a photon is emitted nonetheless, but with a total requisite time on the order of 10⁻⁴ – 10² sec.  In this case the energy of that photon is much diminished and the wave length is expectedly much longer. That in essence is the definition of phosphorescence.

The energy emitted with fluorescence is usually less than the energy of the absorbed photon, which means that the fluorescence spectrum is red-shifted (i.e., longer wavelength, lower energy) relative to the absorption spectrum. This is referred to as the Stokes shift.  There is usually some overlap of the absorption and the emission spectra.  The Point of overlap is precisely that wavelength where both absorption and emission bands occupy the S0V0 to S1V0  transition.  The lower energy of the fluorescent emission can be understood in part by the observation that these photons usually do not decay to the equilibrium ground state.  They might decay only to S0VN (N>0), as already discussed and shown in the Jablonski diagram below. 

Expressed in another way, the probability of an electron returning to a particular vibrational energy level in the equilibrium ground state is similar to the probability of that electron’s occupancy of that ground state position before excitation.  (In both absorption and emission, the probability that an electron in the excited state will return to a particular vibrational energy level in the ground state is proportional to orbital overlap in the respective states.)  Collectively, the above observations explain why the emission spectrum approximates a mirror image of the absorption spectrum.

Another mechanism of radiation-less energy loss is the phenomenon referred to as quenching.  (Collectively, all radiation-less transition pathways compete with fluorescence emission for return of excited state electrons to the ground state.  These will decrease fluorescence quantum yield, which is the ratio of photons emitted to photons absorbed.)  Quenching specifically refers to the radiation-less energy loss that results from a change in orbital population from the lowest vibrational level of the first excited state, S1, to the highest vibrational level of the ground state, S0.  Several types of quenching are recognized in fluorescence spectroscopy. Static and dynamic quenching are most common.  Static quenching results from the formation of a complex between the fluorophore molecule and the quencher.  Dynamic quenching results from the interaction between two light sensitive molecules; such as when a fluorophore transfers energy to an acceptor.  Quenching is an important concept in experimental design as it can confound interpretation if not controlled; or, it can be useful for the interpretation the molecular events occurring in the experiment.

Quenching might arise from a variety of competing intramolecular and intermolecular processes that might dramatically lower or possibly eliminate fluorescent emission.  A common example is observed with the collision of an excited state fluorophore and another (non-fluorescent) molecule in solution, resulting in the return of the fluorophore to its ground state with elimination of the emission spectrum.  Such collisional quenching might occur in labeled macromolecules when a particular moiety, by conformational change collides with the fluorophore while in its excited state.  This would be an example of an intramolecular process.  Intermolecular quenching might occur when an electron deficient moiety of a small organic molecule collides with a macromolecule by diffusion in solution.  The molecular electronic mechanisms might include electron transfer, spin-orbit coupling or intersystem crossing to an excited state triplet.  In molecules such as Aβ one might observe concentration dependent quenching due to both intermolecular and intramolecular events.

Another mechanism, termed static quenching, might occur when the quencher and fluorophore form a reversible complex when the fluorophore is in the ground state; thus, limiting absorption by reducing the population of excitable molecules.  Yet another quench mechanism occurs when a fluorophore in its excited state comes sufficiently close to an acceptor molecule to permit transfer of the fluorophore’s excited state energy; such has been termed dipolar resonance energy transfer, or simply resonance energy transfer (RET).  RET can be used as a “spectroscopic ruler” enabling the study of interactions between cellular compartments, and conformational changes in individual macromolecules.

What chemical structures make good fluorochrome?  A simple answer is that polycyclic aromatic compounds will most likely fluoresce.  Even less complex structures might fluoresce if there is adequate delocalization of π-electrons.  Finding a good fluorochrome is not generally a problem; but with increasing molecular size one might be concerned with potential perturbations of whatever biological or chemical model one is studying.

Fluorochromes can be either intrinsic or extrinsic.  Intrinsic fluorochromes include certain aromatic amino acids, such as tryptophan.  Extrinsic fluorochromes might be bound covalently to a signal or marker molecule of biological interest; or they might bind to molecules of biological interest by virtue of the variety of intermolecular forces known to exist, such as hydrophobic, dipolar, and hydrogen bonding.  Fluorochrome size might also be important depending on the biological system explored.

In our work we have used a variety of fluorophores covalently bound to different sites in the Aβ peptide to assess the ability of our compound library to disrupt the aggregation state of a diverse array of Aβ soluble oligomers.  We have also been able to use to good effect the intrinsic fluorescence of Aβ’s single tryptophan in yet other experimental designs.  Our studies have shown that while some compounds facilitate disaggregation by an initial ‘fraying’ action at the hydrophobic end of the oligomer, thereby facilitating access for intrinsic protease action, others appear to up-modulate certain intrinsic brain proteases.  Plans are in the works also to employ a well-documented library of non-covalent Aβ probes.

There are many literature resources that are freely available to those that wish to further explore fluorescence spectroscopy, the Franck-Condon principle, quenching, fluorophores and their research applications, etc. 

Jablonski Diagram 3.jpg

1. M. Sauer, J. Hofkens, J Enderlein; Basic Principles of Fluorescence
Spectroscopy, Chapter 1, Wiley, 2011
2. J. Lakowicz; Principles of Fluorescence Spectroscopy, Springer 3 rd Ed., 2006

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