Time Lapse Microscopy for Everyone

When I first started out in microscopy, the only way to localize a protein inside a cell was using antibodies. That meant fixing your specimen, going through a complicated series of incubations with blocking agents, primary, and secondary antibodies before you could finally sit down at the microscope to view a still image of your cells. The advent of fluorescent proteins revolutionized that tedious process. Thousands of cell lines and transgenic animals are now routinely used to instantly reveal the expression of a protein simply by looking at it under a fluorescent microscope. And the best thing is: you no longer have to kill your cells first.

More and more, we are using microscopes to observe dynamic phenomena in living cells and tissues such as cytoskeletal dynamics, organelle traffic, and cell migration. Manufacturers have accommodated these developments by offering highly sophisticated incubation equipment for microscopes which maintain temperature, humidity, and CO2 concentration at levels that are optimal for cell survival. They have also built various motorized systems into their stands which minimize exposure to light and maintain focus over the course of an imaging experiment. Such systems are not cheap, however, and, while many shared imaging facilities will offer such systems, it can be hard to get access to them because a single sitting can can last several hours or even days. The good news is that you can often use a fairly simple microscope to do some decent time lapse imaging.

• Inverted microscope with a camera port. It doesn’t have to be a fancy system – a routine tissue culture scope will often do a good job. You may want to upgrade one or two of the objectives depending on the type of imaging you are doing. While inverted scopes are ideal for timelapse imaging because they can accommodate almost any type of dish or flask that your cells might be sitting in, you can also use an upright scope if you have water immersion lenses or if you can mount a coverslip over your live sample. Hanging drop slides, for example, can be used on upright scopes but the small volume of the medium makes them less suited to extended time lapse imaging.
• Camera and software that can be programmed to take images at defined time intervals. You don’t need an expensive imaging software suite for this. Many microscope cameras include free software that can perform basic functions such as image capture at defined time intervals.
• Electronic shutter. Essential for fluorescence timelapse imaging but also highly recommended for making brightfield movies, an electronic shutter will ensure that your sample is not subjected to continuous illumination when you are not actually taking images. A list of reasonably priced shutters along with mounting options for different microscopes can be found here: www.uniblitz.com.
• Trigger-out function. This is needed to trigger the shutter to open and close before and after an image is taken. Many newer microscope cameras have a built-in electronic trigger that will send a TTL pulse before capturing an image. All you need to do in this case is connect the camera to the shutter controller (your camera manufacturer should be able to supply the correct cable). If your camera does not have this feature, look to see if your imaging software has a trigger-out function. If you use software to trigger your shutter you will also need to install a trigger board in your computer.
• A thermally neutral location. The biggest enemy of timelapse imaging is changing of focus due to thermal drift. Expansion and contraction of optical components and the microscope frame can cause your sample to move out of focus with just fractional changes in ambient temperature. To minimize these effects, you will want to place your system as far away as possible from radiators, air conditioning vents, or other objects that can cause changes in temperature. An enclosed area with little air circulation is an ideal spot.
• Optional: Glass bottom dishes. If you need to image structures at a sub-cellular level you will want to use objectives with a decent numerical aperture (0.75 or higher). Such objectives will not work well (if at all) when imaging through the plastic bottom of cell culture dishes. Luckily, several manufacturers offer culture dishes with thin glass (coverslip) bottoms. These are obviously more expensive and fragile than regular culture dishes but well worth it if you want great-looking images and movies. If you decide to stick with standard dishes, make sure your objectives have enough working distance to focus through the plastic base of the dish on to your sample.
• Optional: Heating insert. If your sample is temperature sensitive you may want to consider a heating insert for your stage. Heating inserts come in various shapes and sizes to accommodate different types of dishes and microscope stages. These will keep your sample at a comfortable temperature but they do not, of course, maintain CO2 or pH. Note that if you are imaging with an oil immersion lens you will probably also need an objective heater. A cold objective in contact with your sample is a big heat sink and will negate the effects of a heating insert.

• Turn on all the components of your microscope system at least an hour before you start imaging (preferably two hours or longer). This will allow the microscope frame and optical components to warm up and achieve thermal equilibrium before you start imaging. If your system is not at thermal equilibrium your focus will drift during your experiment.
• If you are not using a heating insert, allow your specimen to cool to the ambient temperature before you place it on the stage.
• To minimize evaporation of the medium and resulting changes in osmolarity, wrap some Parafilm around the edge of the dish or use commercially available lids with gas permeable membranes.
• Find a spot on your sample that you don’t want to image to set up your camera and software.
• If you are doing fluorescence imaging, be prepared for your sample to bleach over the course of the experiment so you may want to start with a higher exposure or gain setting than you normally would.
• If bleaching or phototoxicity is a concern, you can often reduce this by choosing camera settings that minimize exposure times. Experiment with the binning, readout frame, and dynamic range settings of your camera.
• Once you have set everything up, it is often a good idea to do a one or two minute test run and review the resulting movie before you start the actual experiment.