Difference between revisions of "Assignment 5, Part 1: viscosity and diffusivity in glycerol-water mixtures"
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:''Notes'': Fluorescent microspheres have been mixed for you by the instructors into water-glycerin solutions A and B. (a) Vortex the stock Falcon tube, and then (b) transfer the bead suspension into its imaging chamber (consisting of a microscope slide, double-sided tape delimiting a 2-mm channel, and a 22mm x 40mm No. 1.5 coverslip, and sealed at both ends nail polish). | :''Notes'': Fluorescent microspheres have been mixed for you by the instructors into water-glycerin solutions A and B. (a) Vortex the stock Falcon tube, and then (b) transfer the bead suspension into its imaging chamber (consisting of a microscope slide, double-sided tape delimiting a 2-mm channel, and a 22mm x 40mm No. 1.5 coverslip, and sealed at both ends nail polish). | ||
− | :''Tip 1'': | + | :''Tip 1'': Ensure that the focal plane you choose to image is not near the coverslip or the slide. If some particles don't move or hardly move at all, it is likely that they are stuck to the coverslip. Adjust the focus so that you are viewing a plane near the middle of the sample. (A good way to do this is to focus on the top and bottom of the sample chamber and then split the difference.) |
:''Tip 2'': Either limit the ROI to a region with only 3 or 4 particles for a long movie (~30-60s) or use the full FOV for a short time (~10s). Long movies with the whole field of view is a sure way to make MATLAB complain. | :''Tip 2'': Either limit the ROI to a region with only 3 or 4 particles for a long movie (~30-60s) or use the full FOV for a short time (~10s). Long movies with the whole field of view is a sure way to make MATLAB complain. |
Revision as of 17:58, 1 March 2019
This is Part 1 of Assignment 5.
Estimating the diffusion coefficient by tracking suspended microspheres
1. Track some 0.86μm Pink Spherotech polystyrene beads in water-glycerin mixtures (Samples A and B contain 30% and 50% glycerin, respectively).
- Notes: Fluorescent microspheres have been mixed for you by the instructors into water-glycerin solutions A and B. (a) Vortex the stock Falcon tube, and then (b) transfer the bead suspension into its imaging chamber (consisting of a microscope slide, double-sided tape delimiting a 2-mm channel, and a 22mm x 40mm No. 1.5 coverslip, and sealed at both ends nail polish).
- Tip 1: Ensure that the focal plane you choose to image is not near the coverslip or the slide. If some particles don't move or hardly move at all, it is likely that they are stuck to the coverslip. Adjust the focus so that you are viewing a plane near the middle of the sample. (A good way to do this is to focus on the top and bottom of the sample chamber and then split the difference.)
- Tip 2: Either limit the ROI to a region with only 3 or 4 particles for a long movie (~30-60s) or use the full FOV for a short time (~10s). Long movies with the whole field of view is a sure way to make MATLAB complain.
2. Record movies of beads diffusing in the two glycerol solutions and use your newly developed code to estimate the diffusion coefficient of each sample.
- Consider how many particles you should track and for how long. What is the uncertainty in your estimate?
- Calculate the viscosity of the solution and estimate the glycerin/water weight ratio of each solution. (Hint: what is the relationship between diffusion coefficient and viscosity? You may find that this chart is a useful reference.)
Turn in for the viscosity part:
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- Overview
- Part 1: MSD difference tracking and microscope stability
- Part 2: Live cell particle tracking of endocytosed beads
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