Troubleshooting Live Cell Fluorescence Imaging

By on June 23, 2015
Expert Session Summaries

We recently finished our Ask the Expert discussion on Improving Live Cell Fluorescence Imaging. This week we had several interesting questions focused on combating the negative effects of imaging on cell health and viability including looking at media options as a possible solution. Other topics included vessels for imaging, fluorescent fusion proteins and options for evaluating cell viability.

During this Ask the Experts session, we also discussed how Thermo Fisher Scientific has addressed challenges associated with imaging weak fluorophors without damaging the cells, photobleaching, or negatively impacting cell health with a specialized media formulation, FluoroBrite DMEM™. FluoroBrite DMEM is a DMEM-based formulation with background fluorescence that is 90% lower than that emitted by standard phenol red-free DMEM. FluoroBrite DMEM has been designed to enhance the signal-to-noise ration of fluorophors so that researchers can visualize even the weakest fluorescent events in an environment that promotes optimum cell health.

This Ask the Expert session was sponsored by Thermo Fisher Scientific and hosted Virginia Spencer. Dr. Spencer is a R&D scientist with a PhD in biochemistry and epigenetics. She completed a post-doc at Lawrence Berkeley National Lab and has extensive experience in live cell fluorescence imaging. Virginia has been working as an R&D scientist for ThermoFisher Scientific since 2010 and is currently developing products for improving cell culture performance and the live cell imaging experience.

Scientists interested in improving their live cell fluorescence imaging should visit this Ask the Expert session. Below is a sneak peek, for a full transcript of the discussion, please see – Ask the Expert – Improving Live Cell Fluorescence Imaging.

Question:

It seems some damage to the cells, even if it is minor, during imaging is rather common for us. Do you have any recommendations for rehabilitating cells after imaging to minimize damage.

The Answer:

Exposure of cell culture media to fluorescent light can cause the formation of free radicals which can damage cells. I would suggest that you give your cells a fresh supply of new media immediately after imaging in fluorescent light. I would also suggest that you transfer your cells back to a CO2 & 37°C incubator as soon as possible. To help prevent cell damage from occurring, I would recommend using a cell culture medium such as FluoroBrite that has low background fluorescence. This will reduce the need for using high laser light intensities that can harm cells.

Question:

Our fluorescent fusion protein is changing the phenotype. Can we salvage this method or should we try something else?

The Answer:

I would recommend cloning your protein into a TET-inducible vector or a vector with a weaker promoter such as EF-1 alpha. You could then perform clonal selection on a heterogeneous population of cells expressing this construct to identify a clone that expresses lower levels of your fusion protein which do not alter the phenotype of your cells. If you image your cells in a low background medium like FluoroBrite DMEM, you could try reducing the intensity of the light source to minimize any cell damage resulting from excessive excitation of your fusion protein. If neither of these options work, consider validating the findings of your imaging experiments with an alternative non-image-based cell or biochemical assay that doesn’t require exposure to fluorescent light or the expression of a transgene.

Question:

Immediately after imaging our cells are fine and also up to about 12 hours later, then the health seems to rapidly deteriorate. Thoughts?

The Answer:

It is difficult to answer this question without knowing more details about your imaging setup. I will assume that you are imaging your cells in CO2-buffered cell culture media and that the environment is 37C, 5% CO2 and humidified. The gradual reduction in cell health suggests that one of the following may be occurring:

  1. Media evaporation

Placing a vessel of sterile distilled water somewhere within the imaging chamber should help with media evaporation. If you are imaging in a multi-well vessel, I suggest also placing sterile water/PBS in all empty wells surrounding the wells containing your cultures.

  1. Insufficient buffering of the media due to low CO2 levels

The system you are using may not be providing enough CO2 to your cultures for adequate maintenance of long term cell health. To test this, I would suggest placing your imaging culture in a media that contains phenol red and visually monitoring the color of the media over time. A change in the media color from reddish-orange to reddish-purple would indicate that there is likely poor CO2 gas exchange.

  1. Phototoxicity due to light exposure

Some cell types are more sensitive to light exposure than others. It could be that your cells are becoming progressively more damaged with repeated light exposure over time. If this is the case, I would suggest that you image your cells in FluoroBrite DMEM or another media with low background fluorescence. The benefit of working with a low background medium is that you can reduce the intensity of potentially damaging fluorescence light without compromising the signal-to-noise ratio of your fluorophore. Another suggestion would be to give your cells a fresh supply of media prior to the point when you see health begin to deteriorate. This is because fluorescent light can cause the formation of free radicals in cell culture media which can be harmful to cells.

  1. Toxicity due to overexpression of a fluorescently-labeled protein

The over-expression of a fluorescently-labeled protein can sometimes induce phototoxicity. You may want to consider cloning your protein into a TET-inducible vector or a vector with a weaker promoter such as EF-1 alpha. You could then perform clonal selection on a heterogeneous population of cells expressing this construct to identify a clone that expresses lower levels of your fusion protein which do not alter the phenotype of your cells.

We have an excellent team of highly knowledgeable technical service representatives at ThermoFisher Scientific. I suggest you reach out to Tech Services for further guidance if none of the ideas mentioned above helps to address your current problem.

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