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Dear Mariana, Jeremy, Ferhan, and Ben,
Thank you very much for your reply, guidance, and tips! I greatly appreciate it. Sorry for the late responses, a bit occupied with the courses.
So the idea is we would like to replicate the particle tracking experiment kinda like this paper : https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3995753/
And the beads we are using is https://www.thermofisher.com/order/catalog/product/F13080
So for the FRAP experiment with dextran, we already could get some nice result.
But the idea is we wish to know about the microrheology aspect too, thus we use the beads to get another view of how it moves around the gel.
I will also try the tips with agarose immobilization, and let's see how it goes 😊
Many thanks everyone for this help!
Love this community!
Sincerely,
Marco
-----Original Message-----
From: Confocal Microscopy List <[log in to unmask]> On Behalf Of Ben Hibbs
Sent: den 27 augusti 2022 08:28
To: [log in to unmask]
Subject: Re: Particle tracking experiment setup
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Hi Marco,
You may have already done this, but as Mariana mentioned - check the beads and gel first. Check your beads before adding to the gel first to understand the imaging/size. If you need to immobilise the beads, set in agarose (https://pubs.acs.org/doi/abs/10.1021/acs.langmuir.6b00229). Also use controls - image the gel without beads to check level of autofluorescence. You should be able to clearly distinguish the two sizes with a confocal, but make sure you are using appropriately pixel size and objective.
For tracking, there are many options, both commercial and open source (https://imagej.net/plugins/trackmate/). Make sure you are sampling appropriately (spatially and temporally) to accurately track the particles.
As Mariana says, there are many options to improve contrast of polystyrene particles. On a confocal, make sure the illumination is properly aligned for Kohler and adjust the condenser diaphragm appropriately for resolution. Via the eyepiece, you can also try oblique illumination by blocking one side of the illumination or using a separate light source.
Best of luck!
Ben
Ben Hibbs
Klein Australia Pty Ltd
M: +61 407 286 742
E: [log in to unmask]
-----Original Message-----
From: Confocal Microscopy List <[log in to unmask]> On Behalf Of Marco Tjakra
Sent: Friday, 26 August 2022 6:31 PM
To: [log in to unmask]
Subject: Particle tracking experiment setup
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Dear all,
I need to do an experiment about gel diffusivity, and to do that we want to make use of polystyrene beads (0.2um and 1 um) in size to check and compare how they move around the gel.
Unfortunately I could not attach the visual result that I got, problem is I am not sure if this is the right one or not, especially when I compare these 2 sizes, I don't find any differences in term of trajectory.
Furthermore, I was quite confused because I could not see the beads on the eyepiece of microscope, but when I did the live it is there on the screen (Microscope is Zeiss 710 LSM).
Could I have been doing things wrong and these are just autofluorescence artifacts from the gel...
Does anyone has experience about the multiple particle tracking please? It would be great if there is a reference to a protocol which I could learn more.
Thank you very much! Have a pleasant Friday and weekend!
Sincerely,
Marco Tjakra<https://katalog.uu.se/profile/?id=N21-377>* PhD Student [log in to unmask]<mailto:[log in to unmask]>
+46 705143018
COLOTAN Project<https://colotan-etn.eu/> Uppsala University * Department of Pharmacy<https://farmaci.uu.se/> Drug Delivery * Bergström Group<https://farmaci.uu.se/research/drugdelivery/research/bergstrom/membe
rs/>
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