Rotationally Gated Fluorescence: Common Theme Behind Molecular Rotors, Photoactivation and Förster Resonance Energy Transfer (FRET)

About the common features behind the mechanism of molecular rotors, photoswitching of engineered fluorescent proteins (GFPs), and Förster resonance energy transfer (FRET). Besides explining the common mechanism, we also offer a new method for increasing sensitivity of viscosity determination even in living cells, by the combination of polarization energy transfer (polFRET) method and a molecular rotor dye either as an energy donor or acceptor.


Introduction
Viscosity represents the strength of interaction of an object or a molecule with other objects or molecules within immediate vicinity in a solution. Its importance follows from the fact that via its involvement in the lateral and rotational diffusion coefficients it determines the rates of biochemical-biological processes in almost every area in life sciences. From another point of view, it determines also the rates of energy dissipation of moving bodies, molecules.
An interesting feature is that while lateral diffusion depends on the inverse 1st power of the object radius, the rotational one depends on the inverse 3rd power of the radius, i.e. on the volume [1], suggesting a rule -which is a rather general one, having significance also in fluorescence -that by increasing dimensionality of motion, the degrees of freedom for energy dissipation is also increased, exemplified by translation and rotation as 1-and 3-D motions ( Figure  1, Panel A).
Tradional approaches of viscosity measurements in cell biology rest on Fluorescence Recovery After Photobleaching (FRAP) mea surements of translational diffusion and fluorescence or phosphorescence anisotropy measurements of rotational diffusion [2][3][4][5][6]. In FRAP, lateral diffusion is quantified by the speed of reappearance of fluorescence in a target volume after selectively destroying by intense light a portion of fluorophores initially present there [2,3].
In fluorescence anisotropy measurements, rotational diffusion is quantified by the speed of randomization of polarization directions of the emitted photons after excitation by linearly polarized light [4][5][6]. These two approaches, and even their combinations in non-imaging and imaging modes, could be realized equally well in the cytoplasm, on the cell membrane, and in the different intracellular organelles and/or in their membranes [2,6]. Substantial interest has been arisen by a class of special dyes called molecular rotors [1] (Figure 1, Panel B).
These are small compounds having several aromatic rings with substantial rotational freedoms around one or more bonds. They have two excited states: In the Locally Excited One (LE) all their rings are in a single plane leading to high quantum efficiency because of the suppression of rotations. In the other, called "Twisted Internal Charge Transfer" (TICT) state, the molecule assumes a 3-D state instead of the original 2-D one, with one or more rings being rotated out from the LE plane [1,7]. Because the dipole moment of the dye is also increased here, this state has also a higher reactivity with the neighborhood, implying also a more efficient loss of energy and a lower quantum efficiency. The point in this process is that the transition from the 2-D planar state to the 3-D non-planar one is basically dictated by the local viscosity and polarity in the immediate vicinity of the dye, with relative sensitivities to these factors controlled by chemical engineering of side groups. As an extreme case, molecular rotors sensitive to only viscosity can be achieved.
This process can also be envisioned as a special type photo-switching between the high quantum efficiency planar state and the lower quantum efficiency TICT state ( Figure 1, Panel C). The transition to the TICT state may also be followed by a red-shift in the color of emission for TICT state energy gaps slightly lower than in the LE state. Furthermore, fluorescence emission might also completely cease for much lower TICT state energy gaps. Experimentally, molecular rotors can quantify an increasing local viscosity by increasing fluorescence lifetime, quantum efficiency, fluorescence intensity, and a blue-shifted fluorescence color [7,8]. The outstanding feature of molecular rotors distinguishing them from polarization-based sensing of viscosity, is the much higher sensitivity, due to the fact that only the rotation of a subportion of a dye occurring in ~psec time is involved, i.e. due to the much smaller inertia [7].
Rotor dyes can be engineered not only for sensing viscosity, but also for indicating contact points between objects, and probing binding reactions [9].
In their work, H Ying et al. [10] developed a rotor dye by a series of chemical modification of BODIPY for serving two purposes at the same time: The 1st purpose is to enable it for adhering to the vicinal dithiol moieties of endoplasmatic reticulum nascent proteins -via an introduction of an arsenicate moiety -and triggering autophagy of endoplamic reticulum ("reticulophagy") by this binding. The 2nd purpose is the quantitation of this binding via the above "rotor effects". The authors have chosen fluorescence lifetime as the probe parameter, to exclude possible disturbances due to changes in local dye concentrations inherent in intensity measurements. An advantage of the rotor dye is that it is adequate to apply a single emission color channel for quantitating viscosity. The other detection channels might be preserved for other dyes for the localization of rotor dye accumulation e.g. lysotracker, mitotracker for lysosomes and mitochondria, respectively. In the above we saw that excitation induced segmental rotation of certain dyes damped by local viscosity may act as a kind of a photoswitching process.
Actually, the operation of the "official" photoswitching or photoactivation phenomenon taking place in certain engineered Visible Fluorescent Proteins (VFPs) rests on a similar principle [11,12]. The conventional Förster responsible for switching off the donor -and switching on the acceptor -at a given donor-acceptor distance is the relative orientation of the absorption dipole of acceptor and the donor local field at the position of acceptor [13,14].
The functional form of this dependence is conveyed by orientation factor for FRET (k 2 ). At each position around the donor it is generally valid that k 2 is maximal when the acceptor dipole is parallel, and zero when it is perpendicular with the direction of donor local field at that special position. It also can be suspected that by increasing the speed of rotation of either of the donor or acceptor might lead to increased FRET, by the increased frequency of parallel encounters [15,16] (Figure 2). To put it another way, k 2 and FRET efficiency might be increased by reducing local viscosity [15,17]. FRET has already been combined with the rotor dyes for sensing local viscosity [7,18]. The problem of sensing viscosity via rotor intensity is that intensity depends not only on viscosity but also on the concentration of the rotor dye.
In the "ratiometric" arrangement, a rotor dye and a conven-  In the majority of cases the three unit vectors are not in the same plane, but they sample three mutually different spatial directions. In these cases the orientation factor is relatively low (0<k 2 <1, blue shaded area under the p(k 2 ) curve), corresponding to a relatively weak FRET, even for small donor-acceptor distances. Inset B): In a fewer number of cases, the three unit vectors are in the same plane (coplanar). In these cases the orientation factor is higher (1<k 2 <4, orange shaded area under the p(k 2 ) curve), corresponding to higher FRET, even for large donor-acceptor distances. Increasing rotational speed either for the donor of the acceptor the frequency of coplanar encounters of the three vectors also increases leading to a more effective loss of donor's excitation energy via FRET ("rotational gate" mechanism).  [19,20]. Rotor dyes can be used as a donor or acceptor of a FRETpair, in the polFRET measuring scheme, either for a ratiometric calibration of the rotor dye, or for improving sensitivity of the polFRET method for the detection of mobility (viscosity) changes. Vertically polarized light traveling in the x-direction excites the donor-acceptor FRET-pairs on the cell surface at the origin of the xyz coordinate frame. The green and red double-cones coinciding with their apexes indicate the orientational distributions of the donor and acceptor emission dipoles. If we take the orientational distributions for the donor and acceptor the same in the absence of FRET, then the orientational distribution of donor dipoles is always narrower than for the acceptor dipoles, because the hyperpolarizing and depolarizing effects of FRET, respectively. On the donor side there is shorter duration of time for physical rotation and homo-FRET to take place, because of the reduction of lifetime by FRET. On the acceptor side, the acceptor anisotropy is the intensity-weighted average on the directly excited and sensitized emission anisotropies, with the latter one being practically zero. The collecting lens collects all fluorescence in the orange sterical cone from the cell momentarily positioned at its focus. DM dicroic mirror separates the donor and acceptor fluorescences (green, red) and projects them into the polarizing beam splitter cubes PBS1, PBS2, which dissect the beams into the vertically and horizontally polarized components, designated also by vertical and horizontal black double-arrows. The polarized intensity components are detected by photomultipliers (grey cylinders). Scattered exciting light is blocked and the fluorescence channels are further specified by placing band-path filters in front of the detectors (not indicated). From the polarized intensity components first, the total intensities (I 1,tot , I 2,tot ), then the fluorescence anisotropies (r1, r2) are determined for the donor and acceptor as indicated in the yellow box. Then FRET efficiency E is computed from the total intensities (I 1,tot , I 2,tot ). Finally, the method enables correlating donor and anisotropies with the FRET efficiency, i.e. correlating donor and acceptor rotational speeds at different separation distances.

Conclusion
We revealed parallel features behind the working meahanisms of molecular rotors, photoswitching of GFPs, and the FRET process.

Polarisation FRET (polFRET) method is suggested in combination
with a molecular rotor either as a donor or an acceptor for increasing the sensitivity of viscosity measurements in a fluorescent microscope or in a flow cytometer.