IntroductionLive 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]
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 an extracellular matrix (ECM), you could fix, cryopreserve, cryosection and then immunolabel the spheroids. This approach produces beautiful, crisp images but does not provide information on the spheroid 3 dimensional context. If you are wanting to obtain 3D contextual information on spheroids that are embedded in ECM, you can partially dissolve the ECM with PBS-EDTA, smear the spheroids along with the residual ECM onto a slide, fix and then immunolabel them.
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 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. If this is not possible, I would suggest the following:
1. Ensure that the imaging environment has the optimal CO2 concentration and temperature which are required for maintaining good cell health.
2. Use an imaging cell culture medium such as FluoroBrite that has very low background fluorescence but all the necessary nutrients for long-term cell health over hours or days. A low background medium will reduce the need for using high laser light intensities that can harm the cells or alter their behavior.
3. Perform a titration experiment where you expose the cells in your experimental system to a range of fluorescent light levels and/or exposure times and monitor the cell behavior over this range. This is still not ideal but could at least help you to identify the lowest amount of light exposure for imaging your cells, thereby minimizing any negative impact on cell behavior.
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 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.
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.
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: https://www.lifetechnologies.com/order/catalog/product/R37601)
2. For imaging total cells (live and dead) versus dead cells, consider using ReadyProbes® Cell Viability Imaging Kit, Blue/Green (SKU R37609: https://www.lifetechnologies.com/order/catalog/product/R37609
3. For imaging only dead cells, you can use NucGreen® Dead 488 ReadyProbes® Reagent SKU R37109 for green (https://www.lifetechnologies.com/order/catalog/product/R37109) or SKU R37113 for red (https://www.lifetechnologies.com/order/catalog/product/R37113).
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 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.
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.
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 cases.
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. 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.
2. 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.
3. 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.
4. 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.