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The Dilemma of Live Cell Imaging – How to See Something That Does not Like Light

If you remember the 1984 Spielberg movie ‘Gremlins’, then you’ll have a pretty good idea how live cells behave under a fluorescence microscope. They yell ‘bright light!’ and die. Cells don’t like light because they have evolved to live under an epidermal layer or skin, or stay safely hidden inside a dark and warm incubator. They are not used to the heavy load of reactive oxygen species produced by fluorescent light and excited fluorophores. To see something more interesting than your precious few transfected cells shriveling up and detaching, you need to take extra special care of your cells.

For a successful live cell imaging experiment you need to shine as little light on your cells (the fluorescence excitation) as possible and collect as large a fraction of the out-coming light (the fluorescence emission) as possible. The first step is to find a suitable microscope, hopefully a nearby friendly and efficient core facility has one available for you. You can recognize a good live cell scope by looking for a microscope that is inside a large heated acrylic cabinet and has a smaller chamber on the microscope stage which provides 5% humidified CO2 for your cells. There are other ways to do live cell imaging, but this one is the easiest to recognize. If the person (typically it’s the guy with the beard) who is responsible for the microscope starts to happily babble alphabet soup along the lines of EMCCD, sCMOS, LED and speak about band-pass filters, fast shutters and water-immersion objectives when you ask if the system is good for you, you are on the right track.

A typical sample format for live cell imaging is a glass bottom dish which is imaged from underneath on an inverted microscope with a 60x water-immersion objective. If needed, the glass can be coated, with, e.g. fibronectin, to facilitate cell adhesion. Find out what the correct glass thickness for your microscope is and how to use the cover slip correction ring on the objective, they will make a huge difference to the image brightness and sharpness.

Before imaging, wash your cells gently and put them in fresh medium- this helps to decrease the background fluorescence in the media and removes most of the bright dead cells.

My personal rule-of-thumb is to take a maximum of two hundred frames. Which, depending on the experiment, can be taken over 20 seconds or 20 hours or anything in between.

Focus stability is also an important issue- happy and healthy cells are not that interesting to look at if they are blurry. Most of the focus drift is caused by temperature changes in the system. As a rough measure- one degree temperature change will result in a one micrometer focus change. There are three ways to handle this:

1)     Buy a commercial autofocus system.

2)     Keep the temperature in your microscope really stable. This includes putting the sample dish inside the microscope half an hour before imaging it.

3)     Stand beside the microscope and adjust the focus by hand during the imaging.

Think what you are doing and always expose your cells to the least amount of light possible. I have more than once seen a well designed experiment where the total exposure time during a 200 frame time-lapse experiment is well under one minute, but the cells die half-way through because the researcher used the same single cell to set up the experiment and then looked at it by eye for a minute to see whether this really was the good cell. It wasn’t after that.

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