User:Mauricio Bedoya/Notebook/Project 1/2008/05/03

Microrheology properties of Hyaluronan

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First HA experiments 05/02 Added 200ul of buffer to 1mg of HA (5mg/mL) Rehydrated 500nm fluorescent red particles on buffer used 100÷1000ul pippetor 05/03 prepared a sample solution of HA (.5mg/mL) 5ul of HA sample 1ul of beads 94ul of buffer number of particles seems ok in the movie (not too cluttered, but enough) prepared a half concentration solution 50ul of previous soln 50ul of buffer not really, made a mistake with the pipettor and finally had to mix 7.5ul of buffer with 7.5ul of previous soln expA 2000fps 497us of expt 2100 graylevels 90x magnification some tracers stuck to the base of glass expB similar conditions to expA slightly different illumination some aggregates are observed and some dust expC 200fps expD 2000fps Half concentration HA (.25mg/mL) and of beads expE 200fps Half concentration HA and of beads First HA experiments: particle analysis There seems to be a problem with the subpixel resolution, I tried to find a quick fix but couldn't. There is a small valley around 0.5, but is not very deep. Next step ideas 1. Most important for next experiment : use method to filter out particles with sub-pixel resolution, or use 100x objective with 1.5 mag. 2. Particles stuck to the glass are ok - but this reminds me to double check with you that you are studying the particles at quite a distance from the surface (>10 particle radii at least) to avoid hydrodynamic coupling 3. were there any signs of aggregation? Would it make sense to increase the particle density to get better stats? 4. Since the difference in alpha is not significant, lets be dramatic and go to a two concentrations above the critical concentration overlap, say 1mg/mL and 1.5 mg/mL and see what things look like there. At these high concentrations - particle motion might be really small, so use 150x magnification. Be sure to get lots of data at these dilutions, since it is a bit expensive. 5. Lets also test at a concentration that Heike previous had issues with .75 mg/mL and see if aggregation is occuring - and also to get a nice data point. 6. Lengthscale of movies: if you want to have the long time data, it looks like you'll need more stats - you might as well, so long as you have the samples sitting there and no aggregation is occuring. This also might be good for data for two point MR analysis (at a later date). Data Analysis : 1. Can you see any difference in the slope of the two concentrations when you fit it?