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Steady-state acceptor fluorescence anisotropy imaging under evanescent excitation for visualisation of FRET at the plasma membrane.

Devauges V, Matthews DR, Aluko J, Nedbal J, Levitt JA, Poland SP, Coban O, Weitsman G, Monypenny J, Ng T, Ameer-Beg SM - PLoS ONE (2014)

Bottom Line: Higher activity of the probe was found at the cell plasma membrane compared to intracellularly.Imaging fluorescence anisotropy in TIRF allowed clear differentiation of the Raichu-Cdc42 biosensor from negative control mutants.Finally, inhibition of Cdc42 was imaged dynamically in live cells, where we show temporal changes of the activity of the Raichu-Cdc42 biosensor.

View Article: PubMed Central - PubMed

Affiliation: Richard Dimbleby Cancer Research Laboratory, Division of Cancer Studies and Randall Division of Cell and Molecular Biophysics, King's College London, London, United Kingdom.

ABSTRACT
We present a novel imaging system combining total internal reflection fluorescence (TIRF) microscopy with measurement of steady-state acceptor fluorescence anisotropy in order to perform live cell Förster Resonance Energy Transfer (FRET) imaging at the plasma membrane. We compare directly the imaging performance of fluorescence anisotropy resolved TIRF with epifluorescence illumination. The use of high numerical aperture objective for TIRF required correction for induced depolarization factors. This arrangement enabled visualisation of conformational changes of a Raichu-Cdc42 FRET biosensor by measurement of intramolecular FRET between eGFP and mRFP1. Higher activity of the probe was found at the cell plasma membrane compared to intracellularly. Imaging fluorescence anisotropy in TIRF allowed clear differentiation of the Raichu-Cdc42 biosensor from negative control mutants. Finally, inhibition of Cdc42 was imaged dynamically in live cells, where we show temporal changes of the activity of the Raichu-Cdc42 biosensor.

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Related in: MedlinePlus

Steady state fluorescence anisotropy of Rhodamine B solutions of different viscosities for objectives with different NA.(A) Variations of the steady state fluorescence anisotropy of solutions of Rhodamine B with different viscosities for objectives with different NA, Objective 4× (NA = 0.13), Objective 60× (NA = 1.49) in epifluorescence and in TIRF excitation without correcting for the depolarisation induced by the high NA objective (xNA = 2). Fluorescence anisotropy variations with viscosity are shown in (A) and for the different objectives and excitations (B). Each anisotropy value is the average of 5 measurements made on each solution. The average and SD are represented.
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pone-0110695-g002: Steady state fluorescence anisotropy of Rhodamine B solutions of different viscosities for objectives with different NA.(A) Variations of the steady state fluorescence anisotropy of solutions of Rhodamine B with different viscosities for objectives with different NA, Objective 4× (NA = 0.13), Objective 60× (NA = 1.49) in epifluorescence and in TIRF excitation without correcting for the depolarisation induced by the high NA objective (xNA = 2). Fluorescence anisotropy variations with viscosity are shown in (A) and for the different objectives and excitations (B). Each anisotropy value is the average of 5 measurements made on each solution. The average and SD are represented.

Mentions: The fluorescence anisotropy was then measured for the different range of Rhodamine B solutions using equation (3) combined with (4a) and (4b) for the two objectives without correcting in the first instance for the high NA depolarisation (ie xNA = 2). As a reference, Rhodamine B steady state fluorescence anisotropy was also measured using a spectrofluorometer (FluoroMax4, Horiba, UK.). As can be seen on Figure 2.A and Table 1, there is a significant decrease in the measured fluorescence anisotropy in the case of solutions imaged with high NA objective which is more pronounced under TIRF excitation. In fact, in evanescent excitation, since light is predominantly collected from fluorophores in close proximity to the glass surface, their emission properties will be modified by the presence of an interface [84]–[86]. Consequently, the closer the dipoles are to the surface, the more energy is transferred into propagating light for angles higher than the critical angle, resulting in redistribution of intensities. Several studies have shown that a high numerical aperture objective and its ability to “observe” an oriented fluorophores from a continuous range of angles reduce the polarisation of the emission [63]–[68], [87]. Corrections have been derived in order to take into account the depolarisation induced by the high NA [63]. However, these formulas are approximations since they assume that no interface is present and it is known to affect the emission polarization behaviour of a fluorophore. A more detailed calculation was thus undertaken which includes near field coupling effect, taking into account that for dipoles close to a surface, some of the near field energy that doesn't propagate is captured by the surface and converted into propagating energy [86], [87]. Thus, the presence of a dielectric surface perturbs the emission of fluorescence in close proximity and is dependent on fluorophore orientation and proximity to surface. In fact, a theoretical study by Hellen et al.[87] looked at how a bare glass-water interface affects the angle-dependent intensity, collected power, polarization, and lifetime of the fluorescence emission as a function of the orientation and distance of the fluorophore with respect to the interface, looking at the emission properties of fluorophores considered as fixed power dipole. Another theoretical study done by Burghardt et al.[88] by deriving integral expressions for the electric field emitted by an oscillating electric dipole when the dipole is near a dielectric interface, has showed that an interface alters the effective aperture of an objective by reflecting and refracting incident plane waves and it also perturbs the emitted evanescent waves such that some of them become transverse propagating waves with a strong dependence on the distance of the dipole to the interface. More recent works [89] showed that a large part of the radiation of a fluorescing molecule in air or water located in close proximity to a glass surface will be emitted as supercritical angle (SAF) into the glass above the TIR angle. The dependency of the angular radiation pattern of a fluorescing molecule with its distance to the surface can thus be used in order to improve confinement at the detection [90], [91]. Consequently the proximity of the surface is going to affect the emission properties of the dipole and effects are qualitatively similar for both excitation polarization [86]. The presence of the interface and the near field coupling induced under evanescent wave excitation results thus in an increased depolarization effect in TIRF compared to in epifluorescence fluorescence excitation as shown in Figure 2.B. The high NA depolarisation correction factor xNA was determined experimentally as previously described [71], by considering the measurements made with the low NA objective as reference and extracting the correct xNA in order to match them with the measurements made with the 60× objective using the following equation:(5)


Steady-state acceptor fluorescence anisotropy imaging under evanescent excitation for visualisation of FRET at the plasma membrane.

Devauges V, Matthews DR, Aluko J, Nedbal J, Levitt JA, Poland SP, Coban O, Weitsman G, Monypenny J, Ng T, Ameer-Beg SM - PLoS ONE (2014)

Steady state fluorescence anisotropy of Rhodamine B solutions of different viscosities for objectives with different NA.(A) Variations of the steady state fluorescence anisotropy of solutions of Rhodamine B with different viscosities for objectives with different NA, Objective 4× (NA = 0.13), Objective 60× (NA = 1.49) in epifluorescence and in TIRF excitation without correcting for the depolarisation induced by the high NA objective (xNA = 2). Fluorescence anisotropy variations with viscosity are shown in (A) and for the different objectives and excitations (B). Each anisotropy value is the average of 5 measurements made on each solution. The average and SD are represented.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4215982&req=5

pone-0110695-g002: Steady state fluorescence anisotropy of Rhodamine B solutions of different viscosities for objectives with different NA.(A) Variations of the steady state fluorescence anisotropy of solutions of Rhodamine B with different viscosities for objectives with different NA, Objective 4× (NA = 0.13), Objective 60× (NA = 1.49) in epifluorescence and in TIRF excitation without correcting for the depolarisation induced by the high NA objective (xNA = 2). Fluorescence anisotropy variations with viscosity are shown in (A) and for the different objectives and excitations (B). Each anisotropy value is the average of 5 measurements made on each solution. The average and SD are represented.
Mentions: The fluorescence anisotropy was then measured for the different range of Rhodamine B solutions using equation (3) combined with (4a) and (4b) for the two objectives without correcting in the first instance for the high NA depolarisation (ie xNA = 2). As a reference, Rhodamine B steady state fluorescence anisotropy was also measured using a spectrofluorometer (FluoroMax4, Horiba, UK.). As can be seen on Figure 2.A and Table 1, there is a significant decrease in the measured fluorescence anisotropy in the case of solutions imaged with high NA objective which is more pronounced under TIRF excitation. In fact, in evanescent excitation, since light is predominantly collected from fluorophores in close proximity to the glass surface, their emission properties will be modified by the presence of an interface [84]–[86]. Consequently, the closer the dipoles are to the surface, the more energy is transferred into propagating light for angles higher than the critical angle, resulting in redistribution of intensities. Several studies have shown that a high numerical aperture objective and its ability to “observe” an oriented fluorophores from a continuous range of angles reduce the polarisation of the emission [63]–[68], [87]. Corrections have been derived in order to take into account the depolarisation induced by the high NA [63]. However, these formulas are approximations since they assume that no interface is present and it is known to affect the emission polarization behaviour of a fluorophore. A more detailed calculation was thus undertaken which includes near field coupling effect, taking into account that for dipoles close to a surface, some of the near field energy that doesn't propagate is captured by the surface and converted into propagating energy [86], [87]. Thus, the presence of a dielectric surface perturbs the emission of fluorescence in close proximity and is dependent on fluorophore orientation and proximity to surface. In fact, a theoretical study by Hellen et al.[87] looked at how a bare glass-water interface affects the angle-dependent intensity, collected power, polarization, and lifetime of the fluorescence emission as a function of the orientation and distance of the fluorophore with respect to the interface, looking at the emission properties of fluorophores considered as fixed power dipole. Another theoretical study done by Burghardt et al.[88] by deriving integral expressions for the electric field emitted by an oscillating electric dipole when the dipole is near a dielectric interface, has showed that an interface alters the effective aperture of an objective by reflecting and refracting incident plane waves and it also perturbs the emitted evanescent waves such that some of them become transverse propagating waves with a strong dependence on the distance of the dipole to the interface. More recent works [89] showed that a large part of the radiation of a fluorescing molecule in air or water located in close proximity to a glass surface will be emitted as supercritical angle (SAF) into the glass above the TIR angle. The dependency of the angular radiation pattern of a fluorescing molecule with its distance to the surface can thus be used in order to improve confinement at the detection [90], [91]. Consequently the proximity of the surface is going to affect the emission properties of the dipole and effects are qualitatively similar for both excitation polarization [86]. The presence of the interface and the near field coupling induced under evanescent wave excitation results thus in an increased depolarization effect in TIRF compared to in epifluorescence fluorescence excitation as shown in Figure 2.B. The high NA depolarisation correction factor xNA was determined experimentally as previously described [71], by considering the measurements made with the low NA objective as reference and extracting the correct xNA in order to match them with the measurements made with the 60× objective using the following equation:(5)

Bottom Line: Higher activity of the probe was found at the cell plasma membrane compared to intracellularly.Imaging fluorescence anisotropy in TIRF allowed clear differentiation of the Raichu-Cdc42 biosensor from negative control mutants.Finally, inhibition of Cdc42 was imaged dynamically in live cells, where we show temporal changes of the activity of the Raichu-Cdc42 biosensor.

View Article: PubMed Central - PubMed

Affiliation: Richard Dimbleby Cancer Research Laboratory, Division of Cancer Studies and Randall Division of Cell and Molecular Biophysics, King's College London, London, United Kingdom.

ABSTRACT
We present a novel imaging system combining total internal reflection fluorescence (TIRF) microscopy with measurement of steady-state acceptor fluorescence anisotropy in order to perform live cell Förster Resonance Energy Transfer (FRET) imaging at the plasma membrane. We compare directly the imaging performance of fluorescence anisotropy resolved TIRF with epifluorescence illumination. The use of high numerical aperture objective for TIRF required correction for induced depolarization factors. This arrangement enabled visualisation of conformational changes of a Raichu-Cdc42 FRET biosensor by measurement of intramolecular FRET between eGFP and mRFP1. Higher activity of the probe was found at the cell plasma membrane compared to intracellularly. Imaging fluorescence anisotropy in TIRF allowed clear differentiation of the Raichu-Cdc42 biosensor from negative control mutants. Finally, inhibition of Cdc42 was imaged dynamically in live cells, where we show temporal changes of the activity of the Raichu-Cdc42 biosensor.

Show MeSH
Related in: MedlinePlus