If you remember the 1984 Spielberg movie ‘Gremlins’, then you’ll have a pretty good idea of the effect of photodamage in fluorescent live-cell imaging. They yell ‘bright light!’ and die. Cells don’t like light because they have evolved to live under an epidermal layer of skin or stay safely hidden inside a dark and warm incubator.
For a successful live-cell imaging experiment, you need to shine as little light as possible on your cells (the fluorescence excitation) and collect as large a fraction of the out-coming light as you can (the fluorescence emission).
The Problem of Photodamage in Fluorescent Live-Cell Imaging
The high-intensity light that used to excite fluorophores in fluorescent microscopy is toxic to living cells. This toxicity is primarily due to the production of chemically reactive species such as reactive oxygen species.  The cells 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 due to this photodamage, you need to take extra special care of your cells.
Steps to Minimize Photodamage in Fluorescent Live-cell Imaging
Find a Suitable Microscope
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 responsible for the microscope starts to happily babble alphabet soup along the lines of EMCCD, sCMOS, LED, and speaks 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 good microscope will help ensure your cells are as happy as possible during imaging by allowing you to control for other key conditions, such as humidity, CO2 and temperature allowing you to set the optimal conditions for live-cell imaging.
Make Friends with the Core Facility Staff
Or the people responsible for managing and maintaining the microscope you’ll be using. These individuals are worth their weight in gold, as they will have expert knowledge on how to best set up your experiment, troubleshooting issues, and in-depth knowledge of that particular microscope.
Choose the Right Container
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 (e.g.with fibronectin) to facilitate cell adhesion. Find out what the correct glass thickness for your microscope is and how to use the coverslip correction ring on the objective, they will make a huge difference to the image brightness and sharpness.
Wash Your Cells to Remove Any Dead Cells
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.
Limit the Number of Frames You Take
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.
Stay in Focus
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- a one-degree temperature change will result in a one-micrometer focus change. There are three ways to handle this:
- Buy a commercial autofocus system.
- Keep the temperature in your microscope really stable. This includes putting the sample dish inside the microscope half an hour before imaging it.
- Stand beside the microscope and adjust the focus by hand during the imaging.
Use Only the Absolute Minimum Amount of Light Required
At every point, think about 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.
Do you have any other tips for minimizing photodamage in fluorescent live-cell imaging? Leave us a comment below.
- Magidson V, Khodjakov A. Circumventing photodamage in live-cell microscopy. Methods Cell Biol. 2013;114:545-560. doi:10.1016/B978-0-12-407761-4.00023-3
Originally published 31 July 2012, revised and updated 27 October 2020.