Photobleaching in Live Cell Imaging

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April 26, 2018

Photobleaching—light-induced degradation of fluorophores—is a significant problem in live cell imaging. The high-intensity illumination used in live cell imaging can reduce the ability of a fluorophore to fluoresce or can render it completely unable to fluoresce. Fortunately, its deleterious effects can be managed. There are a number of proactive ways that scientists can protect fluorescent proteins and dyes from fading and preserve fluorescence signal, without sacrificing cell health.

How to reduce photobleaching

There are several actions scientists can take to reduce photobleaching when performing live cell imaging. Leanna Ferrand, global product support and applications leader, cellular analysis—genomics & cellular research, at GE Healthcare provides the following suggestions:

Use appropriate current fluorophores and fluorescent proteins that match the excitation and emission specifications of the microscope being used for imaging.
Use as minimal an exposure and light power level as possible.
Avoid lengthy exposure to “wasted” light, such as extended periods of time viewing the sample through the oculars while not collecting data.
When designing the experiment, consider all parameters carefully and minimize to the point of only collecting enough information to answer the question at hand, nothing extra. These parameters include the total time of the experiment, how frequently imaging is done throughout the total time, total number of fluorescent channels used at a time, z-stack heights, etc.

Keep in mind that the longer a researcher wishes to image, the more conservative they will have to be in each of the above parameters to ensure they observe the biology in action, not photobleaching and effects of phototoxicity.

Lisa Stewart, sales & marketing manager at Oxyrase, Inc., also recommends:

Using neutral-density filters to reduce light exposure when setting up for a scan.
Choosing the most photostable dyes for the application.

Reagents that reduce photobleaching

Several companies offer reagents that enable and enhance live cell imaging studies and are designed specifically for use in live cell imaging. Note that antifade mounting media commonly used for the imaging of fixed samples cannot be used for live cell imaging due to their incompatibility with live cells, according to Melissa Rebar, Ph.D., director of marketing at Vector Laboratories.

Vector Laboratories’ VectaCell™ Trolox Antifade Reagent for Live Cell Imaging is an antifading additive that is used to preserve and extend the lifetime of fluorescent signals when imaging live cells. Fluorescent proteins or cellular dyes imaged in live cells are as susceptible to photobleaching as an immunofluorescent signal on a slide.

VectaCell Trolox Antifade Reagent is a stable formulation of a water-soluble form of vitamin E. Its antiphotobleaching and antiblinking properties are a result of the combined action of Trolox and its oxidized form, Trolox quinone. This redox system is effective against different reactive oxygen species, such as singlet oxygen, superoxide anion, or hydrogen peroxide. Rebar notes that Trolox has been shown to have a cytoprotective effect and low cytotoxicity for a number of cell lines,.

Since VectaCell Trolox Antifade Reagent is an antioxidant, the optimal working concentration of this reagent in imaging media will depend on the cell type and the cells’ tolerance to hypoxia. Some optimization by the user may be required.

Oxyrase’s approach to reducing photobleaching and photodamage is based on its unique oxygen removal technology.

OxyFluor™ is an enzyme formulation from the plasma membrane of the bacterium Escherichia coli. These enzymes reduce photobleaching and photodamage in live cell fluorescent imaging applications by removing oxygen and free radicals from the sample without affecting intracellular functions, according to the company.

OxyFluor™ preserves the signals of fluorescently labeled target molecules for longer-term imaging and analysis, even with high-intensity illumination. After 120 exposures using standard time-lapse imaging protocol, samples treated with OxyFluor™ are more than 20% brighter than untreated cells, allowing more time to collect data from imaging experiments, Lisa Stewart says. OxyFluor™ can be added to any cell medium or buffer suitable for fluorescent imaging and has minimal effects on cellular viability and proliferation.

In addition, OxyFluor™ preserves the signals of fluorescent proteins, functional probes like MitoTracker dyes, and counterstains such as Hoescht 33342 for extended imaging protocols and more accurate measurements, Stewart adds. It also provides antifade protection against most fluorescent reagents frequently utilized for live cell fluorescent imaging, including green fluorescent protein (GFP), red fluorescent protein (RFP), LysoTracker®, and CellTracker™.

Thermo Fisher Scientific’s product providing protection from photobleaching for live cell imaging is Prolong™ Live Antifade Reagent, which is based on the Oxyrase antioxidant technology. According to the company, ProLong Live Antifade Reagent has been validated to provide protection for a wide range of organic dyes as well as fluorescent proteins. It has also been rigorously tested and shows little to no measurable effect on cell vitality, proliferation, or incidence of apoptosis for at least 48 hours.

Whether it’s precautionary actions or enabling reagents, if you plan ahead and stick to your plan, you should be able to image longer and avoid photobleaching, while preserving reagent signal and cell health.