Mid-week   Leave a comment

This week I worked on live cell imaging. All I did was image a confluent monolayer of cells continuously for about 5 hours per day. Why did I do this?

First, it’s a way to calibrate the equipment- in order to keep the cells alive, they have to be in tightly controlled conditions. Specifically, they need to be held at 37°C, in 95% relative humidity, with a 5% concentration of CO2 (the air we breathe is around 0.0003%). The tricky one is the CO2- the amount controls the pH of the culture media, and the cells like to be at pH 7.0. Cells are *very* sensitive to changes in pH. Because the cells are alive, they are constantly generating acid which has to be disposed of: your cells do this, too. Your blood (among other things), helps to control the pH your cells experience. Our culture media uses sodium bicarbonate– the same chemical in many antacids- to hold the pH steady. If the cells survive, the equipment is working properly, the incubator is sterile, etc. etc.

But the main reason I did these experiments is because one of the experimental techniques we use is “calcium imaging“. This live-cell technique uses a specific chemical (Fura-2) to measure the amount of calcium present in the cell’s cytosol. If the amount of calcium changes, the cell has activated one or more signal transduction mechanisms- if the amount of calcium goes up, the cell has sensed something, and is reacting to it. Determining *what* the cell sensed, *how* the cell sensed it, and what actions were taken by the cell requires a lot of work. But just knowing that the cell responded to a specific stimulus is very much worth knowing- it tells us something about how cells function.

Anyhow, calcium imaging experiments usually run for 2 or 3 hours- the cells are imaged with a microscope continuously for several hours- and so I need to make sure the cells can survive etc. before I use very expensive chemicals. 1 milligram of Fura-2 costs $200, and it lasts for about 2 months before it goes bad (loses effectiveness).

I initially set my CO2 levels using some culture media- most media has phenol red as a visual pH indicator, but the cells are really sensitive, and since I don’t have a way to measure the CO2 directly, I’m watching my cells react as a way to fine-tune the CO2 level by controlling the flow of gas into the microscope incubator.

The first try was a complete failure: the microscope light was on, really brightly, and the cells immediately went into apoptosis. This is why I often use an EMCCD camera for extended live cell imaging, so the next time I turned the lamp down as low as it could go (and minimized the aperture stop, etc. etc.).

This time the cells survived, but did something really weird: Here’s the first frame (all the images on this post were taken using brightfield imaging):

And here’s a frame taken 10 minutes later (I took a picture every minute)

The cells moved! In fact, they moved *a lot*- here’s the final frame, taken 5 hours after the first:

It’s really striking to watch the time-lapse (I can’t post video here), especially since thse cells went into the differentiated state on July 2nd- they are supposed to sit there and not move. Therefore, this is interesting: why did they move around? Most likely, it’s a problem with the conditions. But I needed to try it again and see if the same thing happens. I checked the filter at very low magnification- the cells appeared as swirls and whorls, like a fingerprint- but the monolayer appeared intact, so the cells may be ok.

One comment about these images- these are all “raw” data. Images that I include in my papers (and this is true universally) represent the *best* possible data. There are ethical guidelines regarding image manipulation in scientific publications, but published images almosrt always look significantly better than what I have here.

The next day, the filter I used was bust- most of the cells had lifted off- apoptosis. So the next filter, I used a much lower magnification to see more of the filter. Using identical conditions from the previous day, I acquired a set of images. Here’s the first frame:

Here’s the final frame:

The cells did not move at all! Except for some blebbing, the cells are perfectly fine. What was different?

Thinking about the moving cells, Although the motion is not uniform, the cells do not appear to be anchored to the filter- they are almost gliding over the surface. That shouldn’t happen- the cells hold onto the filter with integrin, and so the cells, all together and at once, must have let go of the filter. This can happen for several reasons- hypoxia is one. Why would I think hypoxia? Well, I didn’t mention that “the moving cells experiment” was different from the next day- the first time, I added 0.5ml DMEM to the apical side because the high-magnification dipping objective needs a little extra fluid present. 0.5 ml doesn’t seem like a lot, but it increased the fluid level by about 0.3 cm, and oxygen has to diffuse that extra distance to get to the cells, decreasing the amount of oxygen available to the cells, which could lead to hypoxic conditions. The diffusivity of oxygen in water is 2*10^-5 cm^2/s, which gives a diffusion length (in one second) of 800 microns. Adding 0.3 cm of fluid (3000 microns) is almost 4 diffusion lengths, which has a large impact in the amount of oxygen available to the cells.

The monolayer that I used with low magnification- without adding fluid- survived the night, so that at least tells me the cells can survive in the microscope incubator for several hours. So, now we try the high magnification lens again, but without adding additional DMEM. Here’s the first frame:

And here’s the final frame, after 5 hours:

Although there cell movement, it’s significantly less that the first time, and additionally, most of the movement occurred within the first 30 minutes. The media turned a little acidic, so I need to lower the CO2 level a bit and switch to other objective lenses; I have 2 higher-magnification lenses that sit closer to the cells. Using these will mean I can have less fluid present, which will make oxygen more available to the cells, even further reducing the chance of hypoxic conditions developing during an experiment.

Yet another problem (hopefully) solved…


Posted September 1, 2010 by resnicklab in Physiology, Science

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