Figure 1.
Excitation beam path for anisotropy measurement of receptor dimerization.
In EPI illumination (black rays) the excitation beam is perpendicular to the sample. The angle of inclination on the sample can be set by mirror M1. Under a highly inclined illumination mode, the excitation beam (red) is partially back reflected (blue) and can be captured on the diaphragm at different (M1) mirror positions and under p- (a–e) and s-polarizations (a’–e’).
Figure 2.
Steady-state fluorescent anisotropy measurement system.
(P: polarizer, L3: focusing lens, DM: dichroic mirror, OB: microscope objective, S: sample, EF: emission filter, TL: tube lens, D: field stop, L4 and L5: imaging lenses, PB: polarization beam splitter, M2 and M3: mirrors, M4: D-shaped mirror).
Figure 3.
Depolarization introduced by a high NA microscope objective.
3(a) The microscope objective is illuminated by a linearly polarized plane wave and focuses the beam onto the sample. This mode of illumination is used in confocal microscopy. (b) The linearly polarized beam is focused into the back-focal plane of the objective (widefield illumination). The sample is illuminated by a collimated plane wave. The “pupil” polarization states in the image planes are depicted using vertically polarized incident beams. Widefield illumination (b) leads to lower loss of polarization in the illumination field than focused beam illumination (a) as confirmed by optical ray tracing simulations.
Figure 4.
HEK 293T cells labeled by mTFP.
(a) Confocal image of HEK 293T cells heterologously expressing the P2Y1 receptor protein, N-terminally tagged with mTFP1. (b) Normalized excitation and emission conditions used for mTFP1.
Figure 5.
Red-edge excitation suppresses homo-FRET.
(a) Excitation and emission spectra of mTFP1 with the applied excitation changed to the 488 nm laser line and with a narrow-range emission filter. In this situation, the red-edge detection suppresses homo-FRET when the spectral overlap between the excitation and emission spectra of mTFP1 is diminished. (b) Red-edge anisotropy measurement in the TIRF mode shows no loss of anisotropy upon agonist addition, demonstrating that the change of rotational diffusion is negligible, and the anisotropy change is the result of increased homo-FRET upon dimerization.
Figure 6.
Measured anisotropy of ECFP monomers and ECFP-EYFP dimers.
Measurements of anisotropy were made in the EPI or in TIRF mode, in cells expressing ECFP alone (i) or ECFP and EYFP sequences linked by an 18-aminoacid spacer (ii).
Figure 7.
Measured anisotropy in different illumination modes, for analysis receptor assemblies in the cell membrane.
Anisotropy, measured in the TIRF and EPI modes, of mTFP1 alone (i), or in a pair joined by a 20-aminoacid linker (ii), or attached to P2Y1R without (iii), or with (iv) the 10 µM2-MeSATP agonist or the 1 µM P2Y1R-selective antagonist MRS-2500 present (v). The diagrams above the bar plots represent the schematic interpretation of the anisotropy results in the TIRF and EPI illumination modes. (i) The monomeric mTFP1 fluorescent protein, expressed alone, localizes only in the interior of the cell, since it does not translocate into the plasma membrane or form dimers. The measured anisotropy value was found to be independent of the illumination modes. (ii) In control measurements with the mTFP1-20aa-mTFP1 construct, the mTFP1 homo-dimer is intracellularly located and showed much-reduced anisotropy due to homo-FRET between the two linked chromophores, under both illuminations. (iii) The P2Y1R was N-terminally tagged with mTFP1 and expressed. In the EPI mode the intracellular, monomeric mTFP-P2Y1R fusion protein produces the main component of the anisotropy, but in the TIRF mode its observed location is strongly restricted to sites within and near the membrane. (iv) Upon agonist treatment, the labeled P2Y1 receptors readily form dimers in the plasma membrane with almost 100% efficiency. (v) Application of a highly potent P2Y1R selective antagonist, completely reverses the agonist-induced decrease in anisotropy in the TIRF mode, confirming the P2Y1R involvement.