Vesicle Fluctuation and Membrane Elasticity

Sajad Saeedinaeeni

Introduction:

Image 1 A vesicle is a small membrane-enclosed sack that can store or transport substances. For a better understanding of complex behavior of lipid membranes, we have to first make clear its basic mechanical properties. Bending rigidity of membranes in different compositions is one such property. Different techniques for measuring the bending rigidity of membranes have been proposed, amongst which the fluctuation analysis is the most popular. The analysis of shape fluctuation of membranes and vesicles is based on time sequence of snapshots as obtained by optical microscopy. Two important time scales have to be considered for the image acquisition. The first one is the exposure time, also referred to as shutter speed, and the second is the acquisition time. The aim of this experiment is therefore to determine the bending stiffness of the membrane of vesicles . To this end a non intrusive statistical method that correlates the vesicles shape distributions to their elasticity and spontaneous bending is used. The underlying theories are presented in Section 2. Following that and in Section 3 the experimental procedure is explained and Sections 4 and 5 will deal with results and the conclusion of the experiment.

2- Theory:
The position of the vesicle membrane can be described as: = Eq(1)

For shapes close to a sphere, where and average radius and

are the polar and azimuthal angles, R is the . For a quasi-

are the weights of each of the spherical harmonics

spherical vesicle, the dimensionless mean square amplitudes of the spherical harmonic modes have the following behavior:
1

Vesicle Fluctuation and Membrane Elasticity

Sajad Saeedinaeeni = Eq(2)

Where and

is the thermal energy and

introduces the effective tension

is the bending stiffness of the membrane.

In this experiment, we can obtain information from the focal plane. Eq (2) allows us to study the fluctuations of the vesicle by observing just one plane, not requiring a three dimensional measurement. Since the sharpest image corresponds to the equatorial plane of a vesicle, we use only the information from the focal plane by setting the polar angle equal to . The two-dimensional contours are then :

Eq(3) Comparing Eq (3) and Eq (1) for = one finds the following expression for the mean

square amplitudes in the equatorial plane (which here coincides with the focal plane because gravity has no effect on the vesicle shape): = Here is the cut-off of the shortest possible wavelength, Eq(4) is the Legendre polynomial

from the spherical harmonics, and

is a normalization factor.

The bending rigidity can then be expressed as: Eq(5) Where: Eq(6)

Vesicle Fluctuation and Membrane Elasticity

Where Sajad Saeedinaeeni is estimated to be around , however, for moderate tension Eq(6) converges rapidly, making it sufficient to consider a few hundred terms in the sum.

3-Experimental procedure:
For the experiment the vesicles were investigated via a fluorescence microscope. For this they were put between two glass slides and the edges were sealed. For the analysis a factor of 0.162m/pixel had to be taken into account. For each vesicle 500 pictures were taken

and the dependence of the vesicle radius on time and angle was prepared for 100 400 angles. A sample for the investigated pictures is shown in Figure 4. The vesicles were prepared from dimyristoylphosphatidyl-choline (dmpc) and cholesterol (chol). A schematic of stricter of dimyristoyphosphatidylcholine (dmpc) is shown:

Image 2 For this experiment we put the vesicles between two glass slides and seal on the edge. The observations were made via a Fluorescence Microscope.

Vesicle Fluctuation and Membrane Elasticity

Sajad Saeedinaeeni

Image 3

A fluorescence microscope (Image 3) is an optical microscope which is used to study properties of organic or inorganic substances using the phenomena of fluorescence. The most important components of this microscope are the light source, the excitation filter, the dichroic mirror (or dichromatic beam splitter), and the emission filter The filters and the dichroic mirror have to match the spectral excitation and emission characteristics of the fluorophore (fluorescent molecule) used to label the specimen. In this way, the distribution of a single fluorophore (color) is imaged at a time. The excitatory light of a specific wavelength is passed from through an objective lens and then onto the labeled specimen. The emitted light is separated from the excitatory light by a spectral emission filter and focused by the objective. Thereafter it is passed further to the detector. An example for the application of this kind of microscope is to image distributions of proteins or other molecules of interest by applying fluorescent stains to the specimen.

4-Results:
In Table 1 an overview of the resulting bending rigidities is presented. As the naming did not present information about the cholesterol content of the samples, a distinct behavior can only be assumed. Using MatLab the contour length of the vesicle was determined to be L = (38,16 1,2) m. See( Image 4) Sample Bending rigidity kc [1E-19 J]
4

Vesicle Fluctuation and Membrane Elasticity

Sajad Saeedinaeeni dmpc dmpc+chol dmpc+chol2 1,20,5 2,50,8 1,810,31

dmpc+chol3 2,220,72 dopc_group1fits1 0,90,2 1binned dopc_group1fits1 1,140,53 4binned dopc+chol 1,420,42 dopc+chol_group 1,240,64 2fits16binned dopc+chol_group 1,270,53 2fits23binned dopc+chol_group 0,850,32 2fits33binned Table 1

The following image is how MatLab found the contour length and other properties for each of the 10 vesicles:

Vesicle Fluctuation and Membrane Elasticity

Sajad Saeedinaeeni Image 4

5- Conclusion:
Due to the fact that the cholesterol content of the samples remains to be known, the behavior of samples in other experiments to maintain in ours. The behavior of the dopc samples indicates that their bending rigidity is independent of the presence and concentration of cholesterol while the dmpc samples demonstrate a distinct change. According to earlier experiments of other groups the dmpc bending rigidity increases with increasing cholesterol concentration. We therefore assume the dmpc+chol sample to have the highest concentration of cholesterol while the dmpc+chol2 sample seem to have a lesser concentration of cholesterol.

Vesicle Fluctuation and Membrane Elasticity

Sajad Saeedinaeeni

6-References
1. Preparation material. 2. Philippe Meleard, Claire Gerbeaud, Tanja Pott, Laurent Fernandez-Puente, Isak Bivas, Marin D. Mitov, Jean Dufourcq and Pierre Bothorel. Biophysical Journal 72, 26162629. 1997. 3. Hernndez-Machado, F. Campelo and A. Eur. Phys. J. Special Topics 143, 101-108. 2007. 4. Spring KR, Davidson MW. Introduction to Fluorescence Microscopy. 5. Effect of cholesterol on the rigidity of saturated and unsaturated membranes: fluctuation and electrodeformation analysis of giant vesicles Rub_en Serral Graci_a,x Natalya Bezlyepkina,x Roland L. Knorr, Reinhard Lipowsky and Rumiana Dimova 6. Vesicle Fluctuation and Membrane Elasticity, Soft Matter Protocol by Thomas Heyn.