Written By: Heather Brown-Harding, Ph.D.
Live cell imaging is advantageous for research were you may be worried about artifacts of fixation or when you want to measure a phenomenon over time. Live cell imaging is more difficult to achieve than fixed samples because we need to keep the cells live AND happy along with obtaining the images we need. We can reduce artifacts by keeping the cells in a favorable environment and minimizing external stressors. Here are 5 points to keep in mind when setting up your live cell imaging experiment.
1. Environmental controls.
A specific and controlled environment needs to be created for successful live cell imaging. Specifically, with mammalian cells, we need a specific temperature, CO2 concentration, and humidity. The temperature should be 37 degrees Celsius, CO2 should be 5%, and there needs to be enough humidity so that we don’t have evaporation. We are essentially trying to recreate the environment of the incubator for the cell, while we are imaging.
Unfavorable conditions can cause artifacts, or cell death, defeating the purpose of live cell imaging.
There are some tricks that we can use to maintain a favorable environment. Buffers, such as HEPES, can help buffer against CO2 and acidification of the media. Addition of humidity can help minimize evaporation. Evaporation can change the concentration of nutrients or treatments within the media causing unintended changes.
2. Short exposure or low laser, is possible to prevent phototoxicity.
Light causes phototoxicity by interacting with molecular oxygen and creating free radicals. Free radicals, or reactive oxygen species, cause damage to DNA and proteins if high levels are maintained. One way to avoid this is to use longer wavelengths. Longer wavelengths inherently have less energy, so they are not nearly as damaging to a cell. Just like your skin, it’s better to have long wavelengths interacting with your skin than the UV rays.
Sometimes you need to compromise with subpar image quality in order to not bleach or cause cell death, due to phototoxicity. You have sufficient signal for analysis if it at least three times the signal to the background of your image. It may not be pretty, but it is more important that your cells are happy. If you need an ideal image, I generally suggest taking it at a different time than your time course. This way the phototoxicity won’t affect the data you are quantifying.
As a control for phototoxicity, I suggest imaging your probe or unstained cell with the exposure you plan to use and see if any changes occur without the addition of your treatment. This will ensure that the changes that you see with treatment are not due to just the way the cells are being imaged.
3. You can’t use antibodies for labeling.
Antibodies are great because they can be very specific, but they are generally not compatible with live cell imaging. You must use fluorescent proteins and dyes because antibodies can’t penetrate into the cell without permeabilization. Permeabilization would kill the cell you are trying to study because it can no longer maintain homeostasis
Another problem with using antibodies, is that they can activate or block signaling pathways by binding receptors on the membrane.
All dyes may not be suitable for live cell imaging, though. If a dye has not been published for live cell imaging, several tests may be of use. First, combine the dye with a well-known dye and measure co-localization. If that is successful, then dyes need to be examined for phototoxicity. A dye that induces rounding up, or blebbing, is not ideal for live cell imaging. This can easily be observed with DIC or phase contrast imaging.
Fluorescent proteins tags are often a good choice for live cell imaging but can come with their own set of problems. First is the ability to dimerize or oligomerize with other tags. If your protein is supposed to be a monomer, then make sure that you pick a fluorescent protein that is a monomer. DsRed, a common red fluorescent protein, has a tendency to form tetramers in cells. This may affect the location of your protein as well as interfere with the function of your protein due to the large size of the tag. The best practice is to try several fluorescent proteins and try both terminal ends of your protein of interest. Then try the different constructs and see which one works best for your scientific question.
If you want pre-screened fluorescent protein constructs, I would suggest checking if Addgene has it. This is useful because there is already published data on it, so you will know it works.
4. Focus drift due to temperature changes.
According to Boyle’s Law, where PV equals NRT, if the temperature changes and the pressure remains the same then there will be the expansion of volume. This creates small changes in the location of a sample. Even though these changes are small, in the microscopic world it is enough to cause a problem. When the focus is measured in micrometers, these changes can cause the sample to be out of focus.
The best way to prevent this is to allow your microscope enough time to equilibrate to the higher temperature. If possible, allow your imaging system to warm for 30 minutes to an hour. Then move your sample from the incubator to your imaging chamber and let rest for a further 10 minutes. This is generally sufficient to minimize changes due to temperature changes.
Several commercial companies now have an autofocus function to aid with changes during live cell imaging. They work by bouncing far-red light off the interface of the coverslip and the liquid. The machine then makes adjustments for any changes in the coverslip. This keeps the coverslip in focus, but if your sample moves (e.g. rounding up for mitosis) there is nothing the system can do to correct for that.
This is also known as temporal resolution, or how often you need to image to be able to see the event. Some events are very rapid such as calcium bursts. Some are much slower.
For very rapid imaging, phototoxicity is a big concern. If you’re imaging the same spot over and over, the live cell imaging can’t be a long time course because there isn’t a chance for the cell to recover from the photodamage. Rapid imaging can cause photobleaching as well. This needs to be measured in a separate experiment so you can compensate for it during intensity analysis.
Long time courses require cells to rest and recover homeostasis between imaging sessions. So, if you’re doing a 24-hour time course, often you can only image every 10 or 15 minutes.
When planning out the timing of your imaging you need to take into account exposure/scan speed, the number of channels, time for physical movement of any components and if you are taking a z-stack. The time one full set of images can add up quickly if you are doing multiple channels and focal planes.
Performing experiments with just your cells and your probe before you begin can save you a lot of time in the long run and hopefully a few headaches.
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In her free time, she enjoys playing with her cat & dog, trying out new craft ciders and painting.You can find her on twitter (@microscopyEd) a few times of day discussing new imaging techniques with peers.
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