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Temperature and voltage dependent viscosity of the bilayer from FLIM experiments on GUV

Dans le document The DART-Europe E-theses Portal (Page 143-150)

Voltage and temperature

6.5 Temperature and voltage dependent viscosity of the bilayer from FLIM experiments on GUV

To measure the voltage-induced phase transition of the lipid bilayer with the GUV, the viscosity of the GUV will be measured as function of the voltage on the membrane (transmembrane voltage) through the fluorescence lifetime of an

Figure 6.3: The fluorescence of the GUV depends on the polarisation of the laser (first letter, H for horizontal and V for vertical). The second letter is the polarisation of the emitted light received by the detector. The most intense areas on the image HH are located at the upper and lower part of the GUV while in the VV image, they are on the left and on the right. This indicates that the BODIPY is mostly oriented in the plane of the bilayer. The anisotropy can be computed from these images and is represented on Figure6.4.

Figure 6.4: Fluorescence anisotropy on a GUV computed with eq. 6.3. The anisotropy in the central part on the GUV (green) is 0.2. It reaches 0.6 on the sides of the GUV and -0.3 on the top. These measurements are consistent with the transition dipoles of the BODIPY being parallel to the plane of the bilayer.

Figure 6.5: BODIPY used as fluorescent molecule. The red arrow indicates the transition dipole [80].

embedded BODIPY. In liquid phase, the viscosity is smaller than in the gel phase. The fluorescence lifetime of the fluorophore is thus smaller in the liquid phase as seen in Chapter5.

First, the method to apply a transmembrane voltage on the GUV without the possibility to place electrodes on both sides of the lipid bilayer is detailed. The second part here focuses on the results of FLIM measurements on a GUV. This is done at different transmembrane voltages and at 30C, 35C and 44C.

6.5.1 Method to apply a transmembrane voltage and temper-ature on the GUV

Electrodes cannot be placed inside the vesicle but transmembrane voltages can still be applied [109,36,35]. A GUV in water under an electric field accumulates charges on its surface and can be assimilated to an electric capacitor. These charges create in a transmembrane potential. The GUV can be modelled as a hollow dielectric sphere subjected to uniform field E0 (Figure 6.6). A square field being applied on the GUV, the transmembrane potential∆V at position (r,θ) is given by [82]:

∆V = 1.5rcosθE0(1−expτt), (6.7) withtis the duration of the impulsion of the square field andτ is the charging time of this capacitor. τ depends on the geometry and the electric conductances inside (λ1) and outside (λ2) the sphere [82]:

τ = Cmr(λ2+ 2λ1)

1λ2 . (6.8)

Here,λ2= 85µS.cm−11 = 17µS.cm−1 andCm = 0.01 F.m−2(as calculated in Chapter4 and presented on Figure4.5). As a result, τ ≈82µs for a vesicle of 40mum of diameter.

In the experiments, the voltage was applied in a direction parallel to the obser-vation plane. The vesicles were deposited between the gold electrodes of a chip depicted on Figure6.7. The voltage applied on the vesicle is a square voltage at 50 kHz and all mentions of the voltage values in this part are the peak-to-peak voltage applied on the electrodes. The temperature is applied with a hot plate in contact with the sample. It is then measured with a thermocouple in contact with the chip. The measurements presented here are performed on only one vesicle. Figure6.8 presents a phase contrast image and a fluorescent image of this GUV. The diameter of this GUV is 31µm and it is between two electrodes separated by 300 µm. The transmembrane voltage ∆V applied on this GUV as function of the voltage on the electrodesU can be computed using Eq. 6.7:

∆V ≈0.055Ucosθ. (6.9)

It has been showed that when the transmembrane potential is higher than a threshold, the membrane becomes permeable and the model used here to

Figure 6.6: Sketch of a GUV of radius a in water between two electrodes. When a voltage is applied between the electrode, and electric field E0 is created in the water and is applied on the GUV.

compute the transmembrane voltage has to be modified [109]. The threshold for DPPC lipid bilayer is 210-225 mV [165,132]. In the following experiments, the measurements are done at 30C, 35C and 44C. The inter-electrode voltages vary from 0 V to 8 V peak to peak.

6.5.2 FLIM results

The fluorescence decay observed is bi-exponential like i Chapter 5. It is fitted using the equation:

Ae

t

τ1 +Be

t

τ2. (6.10)

The fitting parameters are gathered in Table 6.1 and reveal that the two life-times have comparable statistical weight. The smallest lifelife-times are about 1.7 ns (which corresponds to 120 cP). The largest lifetimes are about 4.5 ns (about 1200 cP). In the following, the pondered averaged lifetimes is considered and they are gathered in Figure 6.9.

These data validate that when the temperature increases, the fluorescence life-time of the BODIPY in the bilayer drops from 4.15 ns at 30C and 35C with 0 V to 3.8 ns. The same effect is observed here when the voltage increases as the lifetime of the GUV at 30C drops from 4.15 ns with no field to 3.65 ns with 8 V. At 35C, the same dependence is observed (4.15 ns with no field and 3.7 ns with 8 V).

Using the calibration from Figure5.4, the fluorescence lifetime from Figure6.10 and eq. 6.9, the viscosity can be computed as a function of the transmembrane

Figure 6.7: Chip to apply voltage on the GUV (right) and close-up on the region used to hold the GUV. The pattern on this chip is in gold and the GUV are put in between the electrodes. The electrodes on the pattern are not always separated by the same distance which allows to have different electric field with the same voltage.

Figure 6.8: Phase contrast image (right) and fluorescence image (left) of the GUV.

It is in a 260 mmol.L−1 glucose solvent with 1 mmol.L−1 of Hepes and is filled with the 240 mmol.L−1 sucrose solution. It is on a glass slide between two gold electrodes separated by 300µm.

Figure 6.9: FLIM images of a GUV at different temperatures and voltages. The voltage indicated here is the peak to peak voltage applied at the contact of the chip. The electrodes are separated by 300 µm. When the temperature increases, the fluorescence lifetime decreases and the same effect is observed when the voltage increases.

Temperature Peak to peak voltage A τ1 B τ2

30C 0 V 3551.28 1.72014 6274.97 4.65224

35C 0 V 3639.58 1.78713 5790.07 4.69298

44C 0 V 4822.38 1.81982 5452.71 4.52057

30C 3 V 3674.42 1.60184 6194.65 4.61938

35C 4 V 4594.06 1.74951 6446.38 4.62137

44C 4 V 7368.93 1.70763 6811.91 4.28419

30C 6 V 4061.63 1.39409 5586.07 4.47237

30C 8 V 5057.46 1.26364 4473.4 4.42373

35C 8 V 6450.91 1.68979 5810.19 4.52032

Table 6.1: Fitting parameters of the fluorescence decay on the GUV.

0 2 4 6 8

Voltage (V) 3.4

3.5 3.6 3.7 3.8 3.9 4 4.1 4.2

Fluorecence lifetime (ns)

30°C 35°C 44°C

Dans le document The DART-Europe E-theses Portal (Page 143-150)