Confocal Imaging of Confined Quiescent and Flowing Colloid-polymer Mixtures

The overall goal of the following experiment is to quantify the changes in confined phase behavior and transport properties of attractive colloidal suspensions using confocal microscopy. This is achieved by formulating colloid polymer mixtures to model attractive colloidal suspensions. The mixtures are imaged and confined in thin wedge cells to show how confinement modifies the structure dynamics and phase behavior of these mixtures.

Next mixtures are imaged as they flow through micro channels to determine the effect of the strength of attraction between the colloids on the confined transport properties of the mixtures. Automated image analysis and particle tracking algorithms can show how quiescent confinement leads to solidification and image correlation. Algorithms can show how increasing strength of attraction leads to increasingly non-Newtonian flow behavior.

The main advantage of this technique or other, other methods like electron microscope and dynamic light scattering is that it allows direct taxes to single particle projectories from which dynamic and structural metrics can be computed for direct comparison to simulations. This standard method can help answer key questions in the field of soft condensed matter physics, such as how suspension self-assemble in the presence of inter particle interactions, or how solid light suspensions yield under an applied force or stress. For this protocol, have prepared PMMA particles that are labeled with a dye consult, a linked reference material in the text protocol for standard preparations.

Make a three to one mixture by weight of broy heane in deco hydro nataline lean next, add tetra butyl ammonium and organic salt to 1.5 millimolar. The solvent matches the refraction index of the particles and partially screens their charge to precisely determine the density of the particles to suspend the particles at 0.1. Volume by volume in the prepared solvent centrifuge, the suspension at 800 G for 75 minutes.

Check that the particles do not sediment after centrifuging. Then to prevent the particles from settling, add either base solvent dropwise to match the solvent density to that of the particles. Next, prepare a 40%suspension of the PMMA particles in the prepared solvent mixture.

Also prepare linear poly cyrene in the solvent mixture at a high concentration. For example, 50 milligrams per milliliter calculated from the molecular weight and radius of gyration of the polystyrene. Using the concentrated PMMA particle suspension and polystyrene solution makes suspensions of the desired concentration of particles with polymers.

Here the suspension contains 15 volume percent of PMMA particles and the polymer concentration ranges from zero to 25 milligrams per milliliter by dispersed suspensions of two differently sized and differently labeled PMMA particles in equal volume. Fractions can also be mixed with the polymer solution. After mixing each suspension, centrifuge them and then add a solvent dropwise to closely match the solvent density to that of the particles.

Lastly, equilibrate all the samples by placing the sealed samples in a Tumblr for at least 24 hours before imaging them. Begin with assembling rectangular chambers from glass cover slips. The microscope slide thickness here is one millimeter, and as thus, the chamber thickness, which permits bulk behavior by the collared polymer mixes.

If a multi height chamber is desired, make thin wedge shaped chambers using a single cover slip spaced on a wedge. The opening angle of the chamber is less than 0.5 degrees and the chamber thickness increases from six microns to one millimeter. This configuration allows the confinement thickness to be continually varied, fabricate the chambers to be placed on an inverted microscope and seal them with a UV curable epoxy resistant to the solvent mixture The microscope used.

Here is a line scanning confocal scan head attached to an inverted microscope with a 100 x oil immersion lens. Having a numerical aperture of 1.4, use the appropriate wavelengths to excite the dyes and take 50 micron square images at 512 pixels square. Start with a one 32nd of a second exposure and then adjust for image quality.

Next, locate the bottom of the chamber particles will be adhered there. Then move the focus to the midpoint of the chamber. Now take about 500 images at a rate of one frame per second at the midplane for 2D analysis.

Then for 3D analysis, take Z stack images with 0.2 micron gaps over 30 microns In any collateral imaging experiment. Optimizing the imaging conditions to obtain high contrast images is critical. Analyze the images using a particle tracking software package.

An algorithm that resolves the particles to 40 or 50 nanometers should be available. Particle tracking is only possible if the particles move less than the inter particle spacing between consecutive frames. To evaluate the particle structure and dynamics, use the following metrics.

A 3D pair correlation function, A 2D or 3D mean squared displacement, and a 2D or 3D self part of the Van Hove correlation function. For this experiment, make a simple flow cell from a square glass capillary 100 microns wide. Attach Teflon tubing and use glass cover slips for structural support.

Next, load the colloid polymer mixture of interest into a glass syringe and attach it to a dispensing system such as a pump. Now mount the setup to the inverted microscope as used in the previous section. The syringe flow cell and outlet should be kept as level as possible.

Set the flow rate according to the colloid polymer mixture. Concentration through square capillaries 200 to 2000 microns per second have been used during flow. Collect 2D images as described in the previous section at different distances from the channel floor from five to 50 microns.

If the particles appear elliptical, increase the acquisition frame rate. Use tracking software and IDL or MATLAB to analyze the flow. Obtaining sub pixel resolution in particle tracking requires careful adjustment of the tracking parameters to yield OIDs that are not biased in a particular direction.

During slow flow. When particles move less than the inter particle distance between frames, trajectories can be obtained using tracking routines in faster flow. Use image correlation to calculate velocity.

Subdivide each image into horizontal images in the direction of the flow. In sequential images, shift the second image to match the first and use the shift distance to calculate the velocity between image pairs or each subdivision of the original images. Make a histogram of all the calculated shift values.

If the values are not strongly peaked, repeat the experiment at a faster frame rate. PMMA particles with the call a diameter of 0.865 microns were suspended at 15%volume by volume in 23.6 milligrams of polymer per milliliter of mix solvents. Structural and dynamic metrics re used to evaluate the images, including pair correlation and mean square displacement.

Changing suspension can change the structure and dynamics of confined suspensions. For example, a by dispersed suspension of 1.48 micron and 0.73 micron particles was formulated particles occupied 15%of the volume by decreasing the confinement thickness. A solid like colloidal gel phase was induced containing both particle types mean square displacement of the large particles decreased as the system was increasingly confined.

Consistent with solidification flow, properties of weekly attractive and strongly attractive particles were compared. Polymer concentration varied from five to 25 milligrams per milliliter. In suspensions, the weekly attractive suspension showed a density profile that varied strongly with position across the channel.

During a flow of 10 microliters per hour, the density profile of the strongly attractive suspension was nearly constant across the channel under the same conditions. Similarly, there were differences in the velocity profiles of these suspensions. Weekly attractive particles were close to the Newtonian prediction for their velocity, whereas strongly attractive particles deviated slightly from the Newtonian prediction for their velocity, especially closer to the sidewall of the channel where Y is zero.

After watching this video, you should have a good understanding of how to use confocal microscopy to investigate phase behavior and flu properties of colloidal suspensions Following this procedure. Other metrics like cluster sizes and fractal dimensions can be calculated from the particle positions to answer additional questions like how confinement modifies the shape of the aggregated clusters.