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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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Acceptor fluorescence anisotropy of MDA-MB231 cells expressing Raichu-Cdc42 biosensors imaged in epifluorescence or TIRF excitation.Fluorescence intensity (A left side) and acceptor fluorescence anisotropy maps (A right side) of live MDA-MB 231 cells transiently expressing Raichu-Cdc42 biosensor imaged at 20°C. Representative histograms of the fluorescence acceptor fluorescence anisotropy of the corresponding cells for both excitations (B). Mean values obtained from these histograms on cells expressing different constructs Raichu-Cdc42 biosensor, Y40C mutant, T17N mutant or Cdc42 imaged respectively in epifluorescence excitation (C) and in TIRF excitation (D). These measurements were made on two independent experiments and are compared with a two-tailed unpaired t-test with 95% confidence intervals (***p<0.001,**p<0.01, *p<0.05, ns non significant). Transfected MDA-MB 231 cells were typically imaged with 200 ms exposure time for the EMCCD and excitation power between 250 µW to 4 mW depending of the expression level of the construct of interest. The scale bar represents 5 µm.
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pone-0110695-g004: Acceptor fluorescence anisotropy of MDA-MB231 cells expressing Raichu-Cdc42 biosensors imaged in epifluorescence or TIRF excitation.Fluorescence intensity (A left side) and acceptor fluorescence anisotropy maps (A right side) of live MDA-MB 231 cells transiently expressing Raichu-Cdc42 biosensor imaged at 20°C. Representative histograms of the fluorescence acceptor fluorescence anisotropy of the corresponding cells for both excitations (B). Mean values obtained from these histograms on cells expressing different constructs Raichu-Cdc42 biosensor, Y40C mutant, T17N mutant or Cdc42 imaged respectively in epifluorescence excitation (C) and in TIRF excitation (D). These measurements were made on two independent experiments and are compared with a two-tailed unpaired t-test with 95% confidence intervals (***p<0.001,**p<0.01, *p<0.05, ns non significant). Transfected MDA-MB 231 cells were typically imaged with 200 ms exposure time for the EMCCD and excitation power between 250 µW to 4 mW depending of the expression level of the construct of interest. The scale bar represents 5 µm.

Mentions: We monitored Raichu-Cdc42 biosensor activity using aaFRET intracellularly as well as at the plasma membrane of cells by switching from epifluorescence to TIRF excitation. Cells expressing the Raichu-Cdc42 FRET biosensor were imaged live at 20°C and 37°C. First experiments carried out at 20°C showed clear differences in the intensity distribution across the image of the Raichu-Cdc42 construct, linked to the confinement of the excitation in TIRF to the plasma membrane. As can be seen in Figure 4.A in the corresponding intensity images, in epifluorescence excitation, out of focus light is also detected which deteriorates the signal-to-noise and contaminates the signal of interest, whereas in TIRF, the labelling of the whole plasma membrane can be efficiently visualized. In addition to this improvement of the signal visualisation, the average steady-state acceptor fluorescence anisotropy differs with the excitation type. A decrease of the acceptor fluorescence anisotropy was highlighted in TIRF compared to epifluorescence illumination as can be seen in the fluorescence anisotropy maps in Figure 4.A and the corresponding histograms of Raichu-Cdc42 fluorescence anisotropy of the same cell imaged with both excitation types (Fig.4.B). This can be related to a higher activity of the Raichu-Cdc42 biosensor occurring at the plasma membrane compared to intracellularly which correlates with the active GTPase being primarily localized on membranes and Cdc42 involvement with cytoskeleton and actin polymerisation [36], [37]. Additionally, TIRF imaging provides good spatial confinement of the excitation and as a result a better signal-to-noise ratio compared to epifluorescence excitation, where fluorescence signal from planes located above or below the imaging plane are also collected as in Figure 4.A in epifluorescence illumination. This increase in signal-to-noise ratio results in a more symmetric distribution of the anisotropy values in TIRF compared to in epifluorescence excitation (Fig. 4.B).


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)

Acceptor fluorescence anisotropy of MDA-MB231 cells expressing Raichu-Cdc42 biosensors imaged in epifluorescence or TIRF excitation.Fluorescence intensity (A left side) and acceptor fluorescence anisotropy maps (A right side) of live MDA-MB 231 cells transiently expressing Raichu-Cdc42 biosensor imaged at 20°C. Representative histograms of the fluorescence acceptor fluorescence anisotropy of the corresponding cells for both excitations (B). Mean values obtained from these histograms on cells expressing different constructs Raichu-Cdc42 biosensor, Y40C mutant, T17N mutant or Cdc42 imaged respectively in epifluorescence excitation (C) and in TIRF excitation (D). These measurements were made on two independent experiments and are compared with a two-tailed unpaired t-test with 95% confidence intervals (***p<0.001,**p<0.01, *p<0.05, ns non significant). Transfected MDA-MB 231 cells were typically imaged with 200 ms exposure time for the EMCCD and excitation power between 250 µW to 4 mW depending of the expression level of the construct of interest. The scale bar represents 5 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0110695-g004: Acceptor fluorescence anisotropy of MDA-MB231 cells expressing Raichu-Cdc42 biosensors imaged in epifluorescence or TIRF excitation.Fluorescence intensity (A left side) and acceptor fluorescence anisotropy maps (A right side) of live MDA-MB 231 cells transiently expressing Raichu-Cdc42 biosensor imaged at 20°C. Representative histograms of the fluorescence acceptor fluorescence anisotropy of the corresponding cells for both excitations (B). Mean values obtained from these histograms on cells expressing different constructs Raichu-Cdc42 biosensor, Y40C mutant, T17N mutant or Cdc42 imaged respectively in epifluorescence excitation (C) and in TIRF excitation (D). These measurements were made on two independent experiments and are compared with a two-tailed unpaired t-test with 95% confidence intervals (***p<0.001,**p<0.01, *p<0.05, ns non significant). Transfected MDA-MB 231 cells were typically imaged with 200 ms exposure time for the EMCCD and excitation power between 250 µW to 4 mW depending of the expression level of the construct of interest. The scale bar represents 5 µm.
Mentions: We monitored Raichu-Cdc42 biosensor activity using aaFRET intracellularly as well as at the plasma membrane of cells by switching from epifluorescence to TIRF excitation. Cells expressing the Raichu-Cdc42 FRET biosensor were imaged live at 20°C and 37°C. First experiments carried out at 20°C showed clear differences in the intensity distribution across the image of the Raichu-Cdc42 construct, linked to the confinement of the excitation in TIRF to the plasma membrane. As can be seen in Figure 4.A in the corresponding intensity images, in epifluorescence excitation, out of focus light is also detected which deteriorates the signal-to-noise and contaminates the signal of interest, whereas in TIRF, the labelling of the whole plasma membrane can be efficiently visualized. In addition to this improvement of the signal visualisation, the average steady-state acceptor fluorescence anisotropy differs with the excitation type. A decrease of the acceptor fluorescence anisotropy was highlighted in TIRF compared to epifluorescence illumination as can be seen in the fluorescence anisotropy maps in Figure 4.A and the corresponding histograms of Raichu-Cdc42 fluorescence anisotropy of the same cell imaged with both excitation types (Fig.4.B). This can be related to a higher activity of the Raichu-Cdc42 biosensor occurring at the plasma membrane compared to intracellularly which correlates with the active GTPase being primarily localized on membranes and Cdc42 involvement with cytoskeleton and actin polymerisation [36], [37]. Additionally, TIRF imaging provides good spatial confinement of the excitation and as a result a better signal-to-noise ratio compared to epifluorescence excitation, where fluorescence signal from planes located above or below the imaging plane are also collected as in Figure 4.A in epifluorescence illumination. This increase in signal-to-noise ratio results in a more symmetric distribution of the anisotropy values in TIRF compared to in epifluorescence excitation (Fig. 4.B).

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