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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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Schematic of the steady-state acceptor fluorescence anisotropy Total Internal Reflection Fluorescence Microscope.OD: Optical density to control the laser power in pupil plane of the objective. λ/2: Half-waveplate in order to change the orientation of the laser output polarisation. L1&L2: form a telescope to enlarge the beam by a factor of 3. L3&L4: form a telescope to enlarge the beam by a factor of 5. Beam expansion by a factor of 15 in order to illuminate the entire field of view. TM: Mirror mounted on a translation stage, which enables to switch from an epifluorescence excitation to a TIRF excitation. L5: Lens to focus in the back focal plane of the objective (60x, NA = 1.49). BS: Beam splitter. NF: Notch filter to avoid any laser contamination. EMCCD: Electron Multiplying Charge Coupled Device.
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pone-0110695-g001: Schematic of the steady-state acceptor fluorescence anisotropy Total Internal Reflection Fluorescence Microscope.OD: Optical density to control the laser power in pupil plane of the objective. λ/2: Half-waveplate in order to change the orientation of the laser output polarisation. L1&L2: form a telescope to enlarge the beam by a factor of 3. L3&L4: form a telescope to enlarge the beam by a factor of 5. Beam expansion by a factor of 15 in order to illuminate the entire field of view. TM: Mirror mounted on a translation stage, which enables to switch from an epifluorescence excitation to a TIRF excitation. L5: Lens to focus in the back focal plane of the objective (60x, NA = 1.49). BS: Beam splitter. NF: Notch filter to avoid any laser contamination. EMCCD: Electron Multiplying Charge Coupled Device.

Mentions: Intramolecular FRET in biosensors occurring at, or just below, the plasma membrane was monitored by steady state acceptor fluorescence anisotropy performed on a TIRF microscope. A 491 nm continuous-wave diode-pumped solid-state laser (Cobolt Calypso, Sweden) was used as the excitation source. This laser emitted linearly polarized light (linear, vertical, >100∶1), and a half-wave plate was used to control the orientation of the polarization at the objective back aperture (Fig. 1).


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)

Schematic of the steady-state acceptor fluorescence anisotropy Total Internal Reflection Fluorescence Microscope.OD: Optical density to control the laser power in pupil plane of the objective. λ/2: Half-waveplate in order to change the orientation of the laser output polarisation. L1&L2: form a telescope to enlarge the beam by a factor of 3. L3&L4: form a telescope to enlarge the beam by a factor of 5. Beam expansion by a factor of 15 in order to illuminate the entire field of view. TM: Mirror mounted on a translation stage, which enables to switch from an epifluorescence excitation to a TIRF excitation. L5: Lens to focus in the back focal plane of the objective (60x, NA = 1.49). BS: Beam splitter. NF: Notch filter to avoid any laser contamination. EMCCD: Electron Multiplying Charge Coupled Device.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0110695-g001: Schematic of the steady-state acceptor fluorescence anisotropy Total Internal Reflection Fluorescence Microscope.OD: Optical density to control the laser power in pupil plane of the objective. λ/2: Half-waveplate in order to change the orientation of the laser output polarisation. L1&L2: form a telescope to enlarge the beam by a factor of 3. L3&L4: form a telescope to enlarge the beam by a factor of 5. Beam expansion by a factor of 15 in order to illuminate the entire field of view. TM: Mirror mounted on a translation stage, which enables to switch from an epifluorescence excitation to a TIRF excitation. L5: Lens to focus in the back focal plane of the objective (60x, NA = 1.49). BS: Beam splitter. NF: Notch filter to avoid any laser contamination. EMCCD: Electron Multiplying Charge Coupled Device.
Mentions: Intramolecular FRET in biosensors occurring at, or just below, the plasma membrane was monitored by steady state acceptor fluorescence anisotropy performed on a TIRF microscope. A 491 nm continuous-wave diode-pumped solid-state laser (Cobolt Calypso, Sweden) was used as the excitation source. This laser emitted linearly polarized light (linear, vertical, >100∶1), and a half-wave plate was used to control the orientation of the polarization at the objective back aperture (Fig. 1).

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