Improving Live Cell Fluorescence Imaging


Live cell fluorescence imaging provides the opportunity to study cellular function, however there can be challenges with the ability to image weak fluorophors without damaging the health of your cells.

During this Ask the Experts session, we will be discussing the challenges associated with imaging weak fluorophors without damaging the cells, photobleaching, or negatively impacting cell health and how Thermo Fisher Scientific has addressed this problem with a specialized media formulation, FluoroBrite DMEM™. FluroBrite 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.

Questions may include:

Questions may include topics such as how to improve the imaging of cells with culture media versus PBS and how you can both culture and image cells in the same media.

Who should visit the session?

  • Are you trying to fluorescently image a difficult to express protein?
  • Are you looking to increase the signal to noise ratio of your live cell imaging assays?
  • Are you changing from media to PBS for your imaging assays and losing precious cells?
  • Are you performing live cell imaging in PBS? Cells changing morphology when not in culture media?
  • When using phenol red-free media for imaging do you still see high background fluorescence?[/two_third]

[This Ask the Expert session is sponsored by Thermo Fisher Scientific and hosted by 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.

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Question 1

What is the best way to image spheroids?

I recommend imaging spheroids by confocal microscopy in a low background fluorescence media such as FluoroBrite. If you are wanting to image spheroids in suspension, you can try coating the surface of the imaging slide or well with a substrate such as poly-lysine that will adhere to the spheroids. If the spheroids are embedded in … Continued

Question 2

I feel that the imaging itself is having an impact on our cell behavior and is skewing the experiment results. How would you set up a control test to verify? And if so, how can I minimize the impact on cell behavior?

This is a tricky question to answer. The experimental results in a fluorescence imaging experiment could be influenced by exposure to the fluorescent light, the imaging environment or the unnatural expression of an exogenous, fluorescently-labeled transgene. To control for the effect of imaging on cell behavior, it would be best to validate the findings of … Continued

Question 3

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.

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 … Continued

Question 4

Are there media components to avoid that could interfere with imaging?

The main media components that interfere with imaging are phenol red and vitamins. Phenol red quenches fluorescence in both the green and red emission channels causing a substantial reduction in the signal-to-noise ratio of a fluorophor. Vitamins autofluoresce particularly in the green channel and this effect will also reduce the signal-to-noise ratio of a fluorophore. … Continued

Question 5

I am looking for a simple way to evaluate cell viability using imaging, can you recommend fluorescent labeling?

Here is a list of some reagents that provide a simple way to evaluate your cells for viability. 1. For imaging of live and dead cells, consider using LIVE/DEAD® Cell Imaging Kit (488/570) (SKU R37601: 2. For imaging total cells (live and dead) versus dead cells, consider using ReadyProbes® Cell Viability Imaging Kit, Blue/Green … Continued

Question 6

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

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 … Continued

Question 7

Are there any special considerations for using the FluoroBrite media? Additional media changes, etc.

FluoroBrite media works just like regular, CO2-buffered DMEM media. Just add the same concentration of FBS and/or any other supplement that you would normally add to regular DMEM and image your cells in a 5% CO2, 37°C and humidified environment. Total: 0 0 0 0

Question 8

What is your preferred vessel for good imaging results?

For high (or even low) resolution images of live cells, I prefer to use chambered coverslips or 35 mm culture dishes with glass bottom inserts. The thickness of the glass coverslip or insert will depend on the quality of image you would like to achieve, however, a No. 1.5 glass insert/coverslip should work in most … Continued

Question 9

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

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. … Continued