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High frame-rate fluorescence confocal angle-resolved linear dichroism microscopy
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Image of FIG. 1.
FIG. 1.

(a) Principle of angle-resolved LD acquisition. An image stack is recorded for various polarization angles α. The plot is an example of dataset extracted at one pixel. (b) The orientational distribution of fluorescent probes that insert perpendicularly to the membrane can be modeled as a 3D cone, defined by two angles ρ and ψ. (c) The case of probes that lie on the membrane surface can be modeled as a pancake-like distribution, defined by two angles ρ and ψ, too. (d) Experimental setup. The inset shows the linear polarization state of the exciting light, making an angle α in the sample plane. HWP: half wave plate, GP: Glan polarizer, EOM: electrooptic modulator, QWP: quarter wave plate, SBC: Soleil-Babinet compensator, BE: beam expander, DM: dichroic mirror, TL: tube lens, EF: emission filter, PCU: polarization control unit; CSU: confocal spinning disk unit.

Image of FIG. 2.
FIG. 2.

(a) Definition of angles Θ and ε used to describe a general elliptical polarization state. (b) Polarimetric analysis of the excitation light after being transmitted through the CSU, as a function of the input linear polarization angle α. Filled markers: before compensation. Empty markers: after compensation.

Image of FIG. 3.
FIG. 3.

Calibration of the excitation polarization angle vs. AO voltage.

Image of FIG. 4.
FIG. 4.

Signal returned by the camera vs. excitation light power for different concentrations of solutions (connected data points) and different gain values. Response is linear below signal value of 6000.

Image of FIG. 5.
FIG. 5.

Camera noise analysis. (a) Relationship between standard deviation σ and mean value ⟨⟩ for the case = 600. Square markers: data, black line: best fit using relationship , with here κ = 1.3. (b) Dependence on the noise factor κ as the function of gain .

Image of FIG. 6.
FIG. 6.

Left: value of ρ (colorscale) as a function of (, ). Dependence is purely azimutal. Right: value of ψ (colorscale) as a function of (, ). Dependence is purely radial. (a) Cone model of Fig. 1(b) , (b) pancake model of Fig. 1(c) .

Image of FIG. 7.
FIG. 7.

Precision analysis. For each set (ρ, ψ) (indicated by a cross, the rectangle represents error bars on ρ (rectangle width is twice the standard deviation on ρ) and ψ (rectangle height is twice the standard deviation on ψ). See text for simulation parameters.

Image of FIG. 8.
FIG. 8.

Results of measurements performed on a GUV labeled with DiIC. (a) Image of the total fluorescence intensity . (b) An example of dataset extracted at the point indicated by a circle in Fig. 8(a) . (c) Experimental composite image showing the mean orientation ρ (indicated by an orientated stick) and the angular aperture ψ (ranging from 0° to 180° and encoded as a color) of the pancake model used to describe the orientation of the fluorophore. The histogram shows the distribution of ψ values. Scale bar is 5 μm. (d) Numerical simulation of data processing for an artificial dataset mimicking a GUV. Parameters are ψ = 72°, = 2000, = 1000 (enhanced online). [URL: http://dx.doi.org/10.1063/1.4807318.1]doi: 10.1063/1.4807318.1.

Image of FIG. 9.
FIG. 9.

Experimental results on COS-7 cells labeled with di-8-ANEPPQ. (a) Fluorescence image. The colorscale indicates the total intensity ∑ ). (b) Composite image summarizes the mean orientation ρ (indicated by an orientated stick) and the angular aperture ψ (ranging from 0° to 180°, encoded as a color). Insets are close-ups. Scale bars are 5 μm.



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752b84549af89a08dbdd7fdb8b9568b5 journal.articlezxybnytfddd
Scitation: High frame-rate fluorescence confocal angle-resolved linear dichroism microscopy