Membrane collapse in culture media
Above video shows the disruption of the membrane from W404 after around 12 hours in the DMEM media (benchtop). The membrane was not treated with UV-Ozone and degraded pretty early as compared to that from W336 which broke at ~28 hrs. The membranes were loaded with 15um beads to check if they were intact and the beads fall off when the membranes break.
Note: W404 is the one which underwent different treatment recently, whereas W336 has been the usual one. Hence, the recent procedure has not helped in stabilizing the membranes in any way.
PS: In the video, it is quite obvious that there are two spots (irregularities) on the membrane which combine as time passes, and then lead to the collapse. As Jim suggests, these might be air bubbles but doesn’t seem likely as there were other samples which didn’t show such pattern but still collapsed near the same time.
Note: W404 is the one which underwent different treatment recently, whereas W336 has been the usual one. Hence, the recent procedure has not helped in stabilizing the membranes in any way.
PS: In the video, it is quite obvious that there are two spots (irregularities) on the membrane which combine as time passes, and then lead to the collapse. As Jim suggests, these might be air bubbles but doesn’t seem likely as there were other samples which didn’t show such pattern but still collapsed near the same time.
This would be a whole lot more useful if the movie could be taken in reflective imaging mode. This is what Joe used to do, and what I do when exposing them to HF for testing. Since the membrane is clear in transmission, we get little info about the membrane itself. In reflection, you can se the membrane clearly, since it has a high refractive index.
There is also a critical need to quantify the stability of the membranes, since we need a way to determine how much the stability is affected by our various treatments. It this TTF (Time to Failure) measure what we should use?
Also – I thought the preliminary indications were that wafers 404 and 403 were behaving BETTER than the previous membranes. Is the data inconsistent or has the media changed? Is this the “standard” test media?
This movie shows the second membrane releasing after the first fails. It seems there is some tension left in the membrane and that the air bubble started a large scale failure. I think its also clear the membranes are loosening their grip on the underlying substrate possibly across the whole surface. Air bubbles over the silicon support might have broken the membrane in the other cases. Working with Joe I noticed that the membranes are major nucleation sites for air bubbles when left on the bench top – kinda like leaving a penny in a cup of water overnight. I think we should de-gas some samples and see if this helps with preservation.
The most important thing right now is high throughput testing of membrane treatments to find the one treatment that prevents discoloration. Obviously time-lapse imaging of individual membrane failure is not high throughput. Membrane failure goes hand-in-hand with discoloration regardless of the mechanism. Noticing discoloration over the chip doesn’t even require a microscope – one can just set up a whole bunch of chips in parallel (including controls) and snap photos. We need to focus on high-throughput discoloration experiments right now. Its called brute force experimentation and every now-and-then it is the right thing to do.
To get a quick turnaround with results, we should be doing discoloration under highly aggressive conditions. I say use DMEM at 37C (this is also highly relevant for cell culture!) and look for discoloration over the chip. Commenting on the presence or absence of beads over the membranes is sufficient to describe membrane integrity at the end of the observation time, but we don’t need to wait for them to break to begin the next study. Such discoloration studies could be done overnight. The algorithm is simple: 1) do overnight study, 2) snap some photos in the morning, 3) think, 4) draw your best conclusion, 5) make a post and see if there is some quick feedback from others, 6) design some further treatments and repeat. This should yield 3-4 data sets per week and should only take 2-3 hours per day of work leaving plenty of time for coursework. This is the first post on this topic in two weeks. Unacceptable.
Right now there is considerable confusion over whether UV/ozone helps and whether the new wafer methods or helping or not. This confusion arrises because people are anxious for data, tests are done, but nothing gets reported except by word-of-mouth.
Chris could you tell me whats the reflective imaging mode? Can we use it along with TC Gui?
This TTF measure can only tell us the relative effect between the different treatments. Its difficult to measure any membrane property once they’ve been soaked in the media. I can’t think of any method to quantify the stability right now. Could you suggest something?
Before, by behaving better I referred to chip discoloration. But looking at the time at which the membrane snaps its obvious that the membrane in 404 is less stable. Yes its the standard DMEM I’ve been using for all the samples.
I should try de-gassing some samples and see if this helps with preservation.
Jim, what is meant by brute force experimentation?
Also, I will carry on the experimentation of 407 samples given by Dave and post soon.
There’s still some problem posting images on the blog.