The explanation of fluorescence self-quenching is derived from the theory of molecular excitonic interactions developed by Michael Kasha and others in the 1960s (e-mail me offline if you want some specific references to this). When the separation of two fluorophores gets down to the level of molecular dimensions (~1 nm), their electronic configurations are no longer separate. The electronic excited states of the monomers merge to form two new excited states, one at higher energy and one at lower energy relative to the original monomer excited state. The energy difference between the two new excited states is referred to as “excitonic splitting”. The two excitonic states represent constructive and destructive combinations of the original monomer state wavefunctions. The molecular geometry of the interaction is critical. The most common case is a face-to-face interaction of the fluorophores, known as an H-dimer. In this case, the absorption transition from the ground state to the lower excitonic excited state is forbidden (i.e. has essentially zero probability of occurrence). Absorption to the higher excitonic state is allowed. Thus a new absorption band is observed at higher energy (shorter wavelength) relative to the monomer absorption band. This is the spectroscopic manifestation of these interactions classically observed for (but not exclusive to) rhodamine dyes. The upper excitonic state rapidly vibrationally relaxes (i.e energy lost as heat) to the lower excitonic state. From there, radiative transition (fluorescence) back to the ground state from the lower excitonic state is forbidden (just as in the absorption case). Instead, the energy is lost via nonradiative internal conversion (heat) back to the ground state or via intersystem crossing to the triplet state. Increased rates of intersystem crossing for H-dimers compared to monomers have been determined experimentally, at least for cyanine dyes. This represents a spectroscopic “double whammy”, since not only do you lose fluorescence but you get increased triplet state generation with all its accompanying photodamage liabilities.
The H-dimer is not the only molecular configuration possible (although it is the most common). The opposite extreme is a head-to-tail arrangement (J-dimer). For J-dimers, the selection rules for transitions between the ground state and the excitonic states are reversed so absorption shifts to longer wavelengths relative to the monomer and fluorescence is observed originating from the lower exitonic state. The classic example of this is the cyanine dye JC-1, widely used as a mitochondrial potential sensor.
Iain Johnson
Molecular Probes/Invitrogen
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