Experimental
3.3 Analysis techniques
Table 3.7: Volumes of dye solution added to the protrusion formation synthesis
dispersions.
Sample Swollen Chlor. cross. seeds Dying solution
(ml) (% wt.) (µl)
Sw. CPS S6 Cl 1 1.5 0.2 3
Sw. CPS S6 Cl 2 1.5 0.1 3
Sw. CPS S7 Cl 1 1.5 2.5 12.9
Sw. CPS S7 Cl 2 1.5 0.1 3
The cell is exposed to a peak to peak current of 4 V (10 Hz) for 2 hours with a gentle linear increase for the first two minutes. In order to prevent attachment to the walls a 5ml glass vial is first filled with BSA solution (5 g/L) once and then rinse with the 0.1 M glucose solution for three times. The GUVs are transferred to this vial using a cut-off pipet to prevent me- chanical stress from destroying the vesicles.
Three experiments 1, 2 & 3 were carried out in order to analyse the effect of crosslinked colloids by surfactant synthesis on the GUVs. For the first and the second experiment the same concentration of colloids was employed but the procedure differed slightly since the tip of the pipet was not introduced in contact with the sample during the second. The different components added in each experiment for the observation of the samples are included in table 3.8.
3.3
Analysis techniques
3.3.1
Bright field and confocal analysis
Bright field and confocal microscopy were used to study the stability and the fluorescence of colloids. For the observation of the samples, both cover
32 Experimental
Table 3.8: Components of each experiment for the observation of the effect of
colloids with SDS on GUVs
Experiment PBS buffer Crosslinked colloids GUVs Con. of colloids
(µl) (µl) (% wt.) (µl) (% wt.)
1 & 2 50 1 4.7 2 0.09
3 50 1 2 2 0.04
slips and capillaries were employed. These can be seen in figure 3.1. In the case of the cover slips, a droplet is deposited on a microscope slide with a Pasteur pipette. Then a cover slip is placed on top and sealed with tape. For the capillaries, they were submerged in the solution until the capillaries were filled. Then the capillaries were glued at the ends to a microscope slide with UV-glue. The glue was hardened by exposing it to UV light for 15 mins. The microscope utilised was a Nikon Eclipse Ti microscope with a 100 x oil objective.
Optical microscopy analysis of GUVs with crosslinked polystyrene col- loids
To study the effect of crosslinked polystyrene colloids stabilised by SDS on GUVs, metal rings holders were used. One of these holders can be seen in figure 3.1. These holders can be unscrewed and a circular glass cover can be slide inside. On top of this cover a teflon inner ring is integrated so that the sample is not in contact with the metal ring. This teflon ring is hermetically contained within the metal ring. Then this cover is rinsed with BSA solution (5 g/L) three times so that adherence of the vesicles to the glass is forestalled. The ring is then filled with 50µl of PBS buffer
100 mOsm (50 mM NaCl), 1 µl of crosslinked polystyrene colloids (0.04
or 0.09 % wt) of batch Cas 2a and 2 µl of the 100 mOsm vesicles. Note
that the vesicles must be the last added using a cut-off pipet and that the concentration of polystyrene is reduced by approximately a factor of fifty in the sample. This sample was then observed with the optical microscope in the bright field and the confocal mode.
32
3.3 Analysis techniques 33
Figure 3.1: Optical microscope sample’s holders left) sample is trapped between
a cover slip sealed with tape and a microscope slide, center) 0.1 x 2 mm capillaries glued with UV-glue to slide right) metal ring holder with inner teflon ring. The width between the two glass interfaces affects the motion of the colloids. That’s why capillaries are used to avoid drift due to evaporation. In the case of the GUVs since they are extremely sensitive to mechanical stress and considerably large, metal ring holders are used.
34 Experimental
Confocal microscopy analysis of Janus particles
When the protrusion is formed on the seeds it is necessary to observe the fluorescence on both the protrusion and the seed after adding the dye. In order to detect that difference some adjustments in the confocal mode of the microscope were made. The galvano mode for confocal microscopy was used with a laser wavelength of 488 nm and a pinhole diameter of 61.3µm. The pixel dwell time was 5.2µs, the image size was 64x64 pixels
and the frame time was 65.1 ms. The reason for these changes was that the fluorescence was very low but photobleaching had to be prevented at the same time.
3.3.2
Scanning electron microscopy analysis
To study the synthesised colloids in detail SEM was used. SEM samples were prepared by washing the colloids at least three times before they were deposited on a SEM stub. To improve conductivity the stubs were sputter coated with Pt/Pd atoms for 200 s at 20 mA at a tilted angle and while rotating.
SEM imaging was performed at a FEI nanoSEM Scanning Electron mi- croscope at 15kV and a spotsize of 3.5 . SEM micrographs were used to study the surface roughness and to determine the size of the colloids. The average size of the colloids was determined manually using ImageJ. For each sample 50-200 colloids were measured to obtain a reliable value for the average size and the corresponding standard deviation.
34