STUDY OF PHOSPHOLIPID LAYERS USING FRAPP SETUP Institut Charles Sadron Supervisors : Thierry Charitat and Pierre Muller In collaboration with Julien Lamolinairie (M2 student) HELSTROFFER SWEN- FESSLER FLORENT M1 Physics - May 2020 N
O INTRODUCTION : Lipid bilayers in biology I T C U D O R T N I
Source : https://www.ck12.org/ Source : Wikipedia
Fundamental constituents Interest for physicists to study their mechanical, of biological membranes. rheological and tribological properties. 2 N
O INTRODUCTION : Long term stakes of the project I T C U D O R
T Understand the tribological and rheological N I properties of phospholipid multilayers
PHOSPHOLIPID MULTILAYERS
We focused on the velocimetry and diffusion parts of the NanoTribo-FRAPP experimental setup in ICS.
3 S
E MATERIALS AND METHODS : I T I L Generalities A R E N E G Hydrophilic head
Hydrophobic tail
Example : the DSPC molecule : Source : Wikipedia Amphiphilic characteristics lead to special conformations when placed in an aqueous medium. (1,2-distearoyl-sn-glycero-3phosphocholine) 4 N MATERIALS AND METHODS : Sample preparation O I T I S
O The deposition of a molecularly thin film on a solid substrate. P
E Depending on the number of dippings, one obtains a succession of layers. D
T T E G Dipper Wilhelmy plate Teflon tank D Movable barrier O L B - R I U M G N A L
Phospholipids Pure water
5 N MATERIALS AND METHODS : Trilayer preparation O I T I S O P E D
T 1 2 3 4 T E G
D Position of the dipper as a function of area Control of the surface pressure O L B - T I U
M 4 G 3 2 N A L
1
Source : L. Fu Phd thesis Source : L. Fu Phd thesis 6 N From sample preparation to FRAPP experiment O I T A R A P
E Know which layer of the R P
system is moving. E L P M A S FLUORESCENT MARKERS PHOSPHOLIPID MULTILAYERS
7 P
U MATERIALS AND METHODS : Principle of the FRAPP setup T E S
P P A R F
How does it work ?
What informations about the sample does it allow to access ?
8 P
U MATERIALS AND METHODS : Principle of the FRAPP setup T E S
P P A R F
Photobleaching of the markers in the sample, following an interference fringe pattern.
Photomultiplier records the contrast between the bleached pattern and a monitoring beam with the same fringe pattern modulated at a certain frequency.
DIFFUSION CONTRAST DECREASE DRIFT CONTRAST MODULATION 9 Y
R MATERIALS AND METHODS : Theory of the FRAPP signal O E H T
L A N G I FLUORESCENCE S
P SIGNAL P A R F
10 Y
R MATERIALS AND METHODS : Theory of the FRAPP signal O E H T
L • All harmonics follow exponential law :
A
N We can get the diffusion coeficient D G
I by fitting harmonics. S
P
P • All harmonics contain a speed
A modulation term R F Access to velocimetry informations.
• A precise oscillation amplitude u of the piezoelectric mirror maximizes each harmonic. Amplitude of 1rst and 2nd harmonics as a function of the ocsillation amplitude of the miror.
11 N RESULTS · Optimization : pockels cell O I T
A Goal: Z I M I Optimize the voltage settings T
P (applied to the pockels cell) to O maximize the ratio : E
S A H P
G N I H
C We recover Malus's Law ! A E L B
Optimal voltage settings : • -10 V for the reading phase. • 10 V for the photobleaching phase. 12 RESULTS · Optimization : mirror amplitude N O I Does the optimization amplitude depend on the interfringe ? T
A How does it behave ? Z
I M I Epoxy resin (with fluo. T
P markers) on microscope slides. O
E D U
T No diffusion of the I
L particles in the sample. P M A
O Probe, for several interfringes, Z E
I which tension amplitude will P optimize the response.
Here : U1 = 1.36 V U2 = 1.70 V U1 U2
13 N RESULTS · Optimization : mirror amplitude O I T
A Optimization of the amplitude as a function of interfringe Z I M I T P O
E D U T I L P M A
O Z E I P
NO CLEAR DEPENDANCE !
14 N RESULTS AND DISCUSSION : Diffusion of polymers in solution O I T
U • Diluted solution of PEG (Polyethylene glycol). L
O By fitting with a characteristic decay time for different interfringes value, one can S recover the diffusion constant of the polymers solution. N I
S R E M Y ) . ) . O U . U . L A A ( P
(
y y t F t i i s s O n
n e e t t N n n i
i O
e I e v S v i i t t a U l a l e F e R F R I D
Time (s) Time (s) Example of 2nd harmonic signals collected on the polymers solution, used to extract a characteristic time : 15 N RESULTS AND DISCUSSION : Diffusion of polymers in solution O I T U
L Diffusion law of a solution of polymers O S If we plug this result in the N I Stokes-Einstein relation : S R E M Y O L
P With :
F The dynamic viscosity (Pa.s) O
Temperature (K) N O
I Blotzmann constant S U F F I D r
16 R
E RESULTS AND DISCUSSION : Study of a DSPC trilayer Y A L I R T
F O
Y D
U We have to perform much T
S longer experiments because of the slow diffusion.
Using this fitting model :
17 R
E RESULTS AND DISCUSSION : Study of a DSPC trilayer Y A L I A priori, there is no macroscopic speed of the sample. R
T Why do we have results as if there was one ?
F O Probably mechanical drifts of the setup ! Y D U T
S Drift of the piezoelectric cell, adding a non constant term in the phase shift ?
Problem due to the heating of the mediums where laser beam passes ?
Symmetrize the 2 beams paths to avoid these problems.
18 N RESULTS AND DISCUSSION : Perspectives and next steps O I T A Z
I • Same experiment but varying humidity and temperature. M I T
P Hermetic box allowing to control O temperature and humidity. • Reach a liquid phase of the bilayer E S instead of a gel phase A H P
G • See how the diffusion constant N I changes according to humidity H
C variations. A E L B
When humidity increases
19 N O I CONCLUSION AND OUTLOOK S U L C N
O The FRAPP setup is a very powerful yet sensitive experimental setup C providing diffusion and velocimetry informations about phospholipid multilayers.
We tried to optimize the FRAPP setup and were able to perform experiments on systems of interest.
Still, we acknowledged the limits and weaknesses of the setup. How to modify the setup in order to avoid mechanical drifts ?
20 Bibliography
[1] Li Fu. Rhéologie des polymères dans les contacts confins: tribologie des interfaces tudies par un nouveau dispositif couplant FRAPP et nano-tribologie. PhD thesis, 2015.
[2] Jean Davoust, Philippe F.Devaux, and Liliane Leger Fringe pattern photobleaching, a new method for the measurement of transport coefficients of biological macromolecules. 1982.
[3] L. Fu, D. Favier, T. Charitat, C. Gauthier, and A. Rubin. A new tribological experimental setup to study confined and sheared monolayers.