Live Cell Imaging and Environmental Control

00:00:11.15 In this tip today, we're going to talk about live cell microscopy
00:00:14.28 in biology, and in particular, the equipment that you need to
00:00:18.10 put on your microscope to keep your cells happy and alive
00:00:21.24 and acting normally while you image them. There are two things you need
00:00:25.20 to consider when you're imaging live cells. The first is just maintaining
00:00:29.05 the environment around the cells so that they grow normally.
00:00:31.27 And for mammalian cells, that means keeping them at 37C
00:00:35.19 and providing CO2. For other cell types, it may mean keeping them
00:00:40.02 at different temperatures. And it also means if you're growing your cells in
00:00:43.26 an open dish, providing a humidified environment so that the
00:00:46.23 media they're growing in doesn't evaporate. There are number of different
00:00:49.28 solutions to this problem for different cell types. For mammalian cells,
00:00:53.11 traditionally we grow cells in liquid media, keep them at 37C, and
00:00:57.27 provide a humidified environment with 5% CO2 to maintain buffering.
00:01:01.04 For cells like yeast cells or bacteria, we often grow them on agarose
00:01:06.27 pads or media included in the pad, so it's just one 1% or 2% agarose
00:01:12.21 dissolved in culture media and solidified, with the cells placed
00:01:17.04 on top. And other cells may require different imaging conditions.
00:01:21.00 Today I'm going to use mammalian cells as an example, because they're
00:01:25.08 the most commonly imaged cell type. So if you look on the scope here,
00:01:29.14 you'll notice that it has this large plexiglass enclosure around it.
00:01:32.23 The scope itself is a normal epifluorescence microscope, but we've built
00:01:38.20 this plexiglass enclosure around it. And the plexiglass enclosure
00:01:41.23 is maintained at 37C to keep both the cells and the entire microscope
00:01:47.09 at 37C. There are air feeds here at the top and the bottom, so these
00:01:53.26 hoses here bring in warm air, and the ones on top extract the cool air,
00:01:58.12 and they recirculate it through this temperature control unit in the back.
00:02:02.09 And that maintains this whole enclosure at 37C within about a tenth of a
00:02:07.20 degree, once it's at steady state. The reason why we enclose the whole
00:02:12.06 scope and not just the sample is that enclosing the scope, as well as
00:02:15.28 the sample helps minimize the effects of temperature fluctuations
00:02:19.05 on focus. So if we didn't enclose the whole scope, we would have a
00:02:23.21 warm sample and a cool scope, and heat transfer from the sample
00:02:27.02 to the scope would cause parts of the scope to expand and contract
00:02:30.07 as the temperature changed, and that would cause the focus to drift.
00:02:33.07 So these large plexiglass enclosures, while cumbersome, provide a
00:02:37.11 very stable temperature controlled environment around the scope
00:02:40.10 and not only keeps our cells at the right temperature, but also
00:02:42.25 minimizes temperature fluctuations that would cause focus drift on the
00:02:45.24 microscope. We'll talk later about other approaches when you don't
00:02:50.06 want this large temperature enclosure around your scope.
00:02:53.04 So that's one key thing in keeping our cells happy, is keeping them
00:02:57.17 at the right temperature. And plexiglass enclosure does that. Right now it's at
00:03:02.00 37, but the controller can be adjusted to any temperature between room
00:03:05.08 temperature and 37C for other cell types. The second part of keeping our cells
00:03:10.09 happy is providing the right environment around them. And
00:03:13.29 to do that, we have a.. basically an enclosure that goes just
00:03:20.02 around the sample. So we have this enclosure here which would
00:03:23.26 sit on top of the sample on the microscope, like so, and then in
00:03:29.19 operation, this would be perfused with air containing 5% CO2
00:03:33.11 to provide the buffering capacity for the cells and also, kept at
00:03:38.00 100% humidity to keep the medium from evaporating.
00:03:40.23 That gas stream is prepared by a gas mixer, up here, where we
00:03:46.19 mix 100% CO2, pure CO2, with room air to make 5% CO2.
00:03:52.29 That 5% CO2 stream then comes into the scope enclosure through
00:03:57.26 this tube here, there is then a humidifying device here, so this is a tank full of
00:04:03.20 water with a specialized hose in it that has very efficient water transfer across
00:04:08.23 the hose, so that the gas coming in is dry and the gas exiting
00:04:12.17 is water saturated. And then that humidified 5% CO2 stream flows into the
00:04:17.23 enclosure here on the scope, where it bathes the cells in that humidified
00:04:21.05 5% CO2 atmosphere. You can do variations on this for hypoxic
00:04:27.24 culture of cells, you can reduce the oxygen, you can use other gas mixtures,
00:04:31.01 but this setup that I'm describing here is both the simplest and the most common
00:04:35.22 setup you'd see on a microscope like this. And it works for
00:04:39.00 most routine cell culture. If you're not doing mammalian cells, generally you
00:04:43.05 don't need the special gas perfusion, you can just use the air,
00:04:47.14 humidified air. You can also use other mechanisms to humidify the air,
00:04:53.04 instead of using this gas permeable hose, you could use a simple
00:04:56.05 bubbler, it's not as efficient but works for simpler culture systems.
00:05:01.13 So that's the purpose of this enclosure here. It's designed to hold
00:05:05.27 cells in their physiological environment at 37C, 5% CO2, and
00:05:11.14 prevent evaporation of the media. That's one thing you need to do to keep
00:05:17.17 your cells happy while you're imaging them live, and is kind of a prerequisite to doing any
00:05:22.22 imaging. When you're doing imaging, there's additional concerns
00:05:26.23 which are beyond the scope of this lecture, but you should be aware of.
00:05:31.09 And those relate to phototoxicity, so the act of illuminating cells with
00:05:36.27 light to excite fluorescence in them is toxic to the cell. And so if you expose your
00:05:41.19 cells to too much light, they will die or undergo stress and will not behave
00:05:47.24 normally. And this can be observed by cells which fail to divide
00:05:52.00 under illumination, cells that stop moving, membrane blebbing,
00:05:56.04 mitochondrial fragmentation, and other cellular pathologies that are the result
00:06:01.16 of light stress from imaging cells. So a second large concern in keeping
00:06:06.23 cells happy and alive while you image them is not illuminating them excessively.
00:06:11.11 And that can mean using low intensity light and short exposures,
00:06:14.26 or infrequent exposures in time lapse to make sure that the cumulative
00:06:18.18 stress from illuminating the cells is not so great that it causes
00:06:21.25 pathological behaviors while you're trying to image them. On this system,
00:06:27.07 we have a much simpler way for doing temperature control for live cell
00:06:31.24 imaging. It's not as robust or as accurate as the plexiglass enclosure
00:06:35.29 on our other microscope, but it is much simpler and it's portable, so we can
00:06:40.07 move it from microscope to microscope. You wouldn't want to use this kind of setup
00:06:44.02 for a long term cell culture, but if you only need to keep your cells alive
00:06:47.20 for an hour or two while you image them, it's perfectly fine.
00:06:51.13 The way this system works is we have two pieces, we have
00:06:55.07 a hot air blower, which blows warm air over the sample, this is essentially
00:07:00.10 a fancy hair dryer with temperature feedback to hold the air stream at a constant
00:07:05.10 temperature. And that will heat the area around the sample generally,
00:07:10.23 and then we have an objective heater, which heats the objective.
00:07:16.01 And so an oil immersion objective like this one, when you have
00:07:21.02 it in contact with your sample, an oil between the top of this
00:07:23.27 objective and the coverslip of the sample, this effectively is a very
00:07:28.01 large heatsink, and it sucks the heat away from your sample
00:07:31.16 very efficiently. So if you don't keep this thermostated, if you don't keep this at
00:07:36.20 37C as well, your sample is not going to be maintained at 37, despite
00:07:41.11 the fact that we have this hot air blower blowing air across the
00:07:43.25 sample. So the way this works, the way this objective heater works,
00:07:48.00 is we've got a little heating coil here that's this top piece here,
00:07:53.03 which just bolts around the top of the objective. So this slides on
00:07:57.12 like so. We tighten it into place. The heating coil here is tightly
00:08:03.26 butted up against the objective and that holds the temperature of the objective
00:08:07.28 at whatever the temperature of this heating coil is set to.
00:08:11.08 So once we do this, we can maintain our objective at 37C, we can use our
00:08:15.06 hot air blower to maintain our sample surrounded at 37C, and that will keep our
00:08:18.26 sample at 37C and keep it warm. On here, we're not providing
00:08:23.11 humidified air or CO2, so this is not an acceptable system
00:08:28.17 for maintaining cells alive in the long term, but for short term
00:08:31.23 imaging for an hour or two, it works fine. And it has the big
00:08:35.08 advantage that unlike the previous system, it's portable from
00:08:37.28 microscope to microscope, so we can use it on any microscope we need.
00:08:41.03 One downside to this system is of course, because it relies on the objective
00:08:46.05 heater here, it only works on a single objective. So we can't
00:08:49.23 switch objectives easily while we're imaging our sample.
00:08:53.19 But with those caveats in mind, this is a cost effective and simple
00:08:57.23 solution for maintaining live cells at temperature and getting good
00:09:01.23 microscopy images of them.

This Talk

Speaker: Kurt Thorn

Audience:

Researcher

Recorded: April 2013

Talk Overview

To image living cells with a microscope, they must stay alive. Here we discuss how to control the temperature, humidity, and atmosphere (CO2 concentration) around your cells so that they survive during live cell imaging. We discuss both simple and more complex systems for environmental control.

Speaker Bio

Kurt Thorn

Kurt Thorn is an Assistant Professor of Biochemistry and Biophysics at UCSF and Director of the Nikon Imaging Center – a facility that provides cutting edge light microscopy equipment to UCSF researchers. Kurt can be followed on his blog at http://nic.ucsf.edu/blog/. Continue Reading