Difference between revisions of "Assignment 5, Part 1: viscosity and diffusivity in glycerol-water mixtures"
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[[Image: 20.309_130924_GlycerolChamber.png|right|thumb|200px|Imaging chamber for fluorescent microspheres diffusing in water:glycerol mixtures]] | [[Image: 20.309_130924_GlycerolChamber.png|right|thumb|200px|Imaging chamber for fluorescent microspheres diffusing in water:glycerol mixtures]] | ||
− | 1. Track some 0. | + | 1. Track some 0.84μm Pink Spherotech polystyrene beads in water-glycerin mixtures (Samples A and B contain 80% and 85% 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). | :''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). |
Revision as of 16:56, 25 February 2020
This is Part 1 of Assignment 5.
Estimating the diffusion coefficient by tracking suspended microspheres
1. Track some 0.84μm Pink Spherotech polystyrene beads in water-glycerin mixtures (Samples A and B contain 80% and 85% 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: Each full frame of the full camera field of view takes up almost 2.5 MB of memory, so movies can get large very fast. Try to limit image data variables to a reasonable size by keeping the length of the movie short or limiting the Region of Interest (ROI) to a fraction of the full field of view.( A full-field, three minute move takes up about 4 GB, which is certain to push MATLAB over the edge.)
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 factors determine the uncertainty and accuracy of your estimate?
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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