Features and analysis of photoenhancement

During an MST experiment, the first few seconds of acquisition are used to define the fluorescence value of the “cold area” for calculation of the MST signal. Thus, the LED excites the sample, but the MST power is still off. During this initial illumination of the sample, most likely the fluorescence will remain constant, whereas for non-photostable dyes the fluorescence might decrease due to photobleaching. However, some experiments exhibit an unexpected behavior, referred to as photoenhancement, where the fluorescence intensity increases upon illumination of the sample. This can even happen in a ligand-dependent manner (see Figure 1). Datasets with ligand-dependent photoenhancement should be analyzed according to their initial fluorescence using the expert mode Bleaching Rate in the MO.Affinity Analysis software to derive binding affinities. MST analysis after the IR laser is switched on should not be considered for affinity determination as fluorescence changes will disturb the signal.

Figure 1: Typical MST experiment displaying ligand-dependent photoenhancement upon illumination. The fluorescence signal displaying the photoenhancement is highlighted in red.

Principle of fluorescence

Fluorescence is the physical property of some molecules (so-called fluorophores) to be able to absorb a photon and emit a photon of lower energy in return. Upon photon absorption, the energy level of the fluorophore changes from the ground state (S₀) to an excited state (S₁). From this excited state, the molecule will return spontaneously to the ground state, emitting a photon. However, after several cycles of fluorescence the fluorophore can bleach. The bleaching is due to the transition from the excited state S₁ to the triplet state T₁(Figure 2), which interrupts the excitation/emission cycle necessary for fluorescence. This means the fluorophore can’t absorb light anymore, thus losing its fluorescence properties. Each fluorophore has its own lifetime before bleaching.

Figure 2: Jabłoński diagram representing the ground state S₀ and singlet state S₁ associated with the excitation by a photon of energy hν_exc and the emission of a photon hν_em during the fluorescence cycle. The black dashed arrow corresponds to the internal conversion prior to photon emission. The black arrow illustrates intersystem crossing resulting in bleaching where the fluorophore goes from the excited singlet state S₁ to the triplet state T₁.

A possible explanation for photoenhancement

In some specific conditions, energy can transfer from the excited state S₁ to a neighboring molecule in a non-radiative way. This phenomenon is referred to as Förster Resonance Energy Transfer (FRET). The most common application of FRET is to study the proximity between two fluorescently labeled molecules by exciting one (the donor) and analyzing the emission of the second one (the acceptor). However, on devices such as the Monolith instruments, where only one type of fluorophore is monitored, such a phenomenon can appear as a decrease in fluorescence intensity of the donor (i.e. quenching). Quenching is defined as the inhibition of fluorescence emission upon excitation of a fluorophore. However, quenching by FRET relies on a photostable acceptor. Therefore, if the acceptor undergoes bleaching, the fluorescence of the donor is restored (Figure 3).

Figure 3: Jabłoński diagram of two fluorophores that are FRET compatible. The fluorophore used for the MST experiment is called “Target” and only two of its energy states, S₀ and S₁, are presented. The ligand inducing the quenching is called “Ligand” and its three different energy states are displayed: two singlet states A₀ and A₁ as well as its triplet state T₁. The FRET between the target and the ligand is represented by the black arrow.

During an MST experiment the labeled target molecule is a photostable donor. If the ligand behaves as a non-photostable acceptor, the ligand will become bleached over time upon continuous illumination. This allows the labeled molecule to emit more light and is one reason for photoenhancement in MST experiments.

Recommendation

To discriminate between binding specific and unspecific effects on photoenhancement, it is recommended to repeat the binding experiment with a negative control target (a non-binding target, such as binding impaired mutants or unrelated proteins, or fluorophore only). In this control, the ligand does not bind the target. If it still changes photoenhancement behavior, the effect on photoenhancement rate is unspecific. If there is no more effect on photoenhancement behavior, the effect is binding-specific and affinity information can be extracted from the photoenhancement rate.