We recently finished our Ask the Expert discussion on fluorescence detection in living cells. The result was an interesting discussion about using cell imaging to evaluate cell viability and improve production. Other topics included methods for live cell imaging, molecules in media, genetic expression of fluorescent proteins and label-free imaging.
Fluorescence imaging of living cells can provide important data regarding the function and localization of proteins and other bio-molecules within a cell or tissue. These images give insight into fundamentally important biological processes and improve our knowledge about transient interactions we might not be able to detect otherwise. An added benefit is some simply remarkable pictures of colorized cells which are just fun to look at.
This Ask the Expert Session was Sponsored by Life Technologies and hosted by Timothy Fawcett, Ph.D. Dr. Fawcett has been in the biotechnology business for over 30 years. Trained as a biochemist he has held senior positions in both academics and industry and has been a mentor to many young scientists throughout his career. For the last 12 years Dr. Fawcett has been the Director of the BioTechnical Institute of Maryland (BTI) a non-profit institute located in Baltimore, Maryland. He is also the Founder and Director of BioSciConcepts, a social venture of BTI that provides hands-on training for professional scientists in cell culture, baculovirus based expression, as well as topics such as molecular biology, PCR and real-time PCR. BioSciConcepts is an internationally recognized provider of expertise in the biological sciences and has provided consultation services to several small and large biotechnology companies.
Below is a sneak peek of the discussion. For a full transcript of the discussion, please see – Ask the Expert -fluorescence detection in living cells.
How can we use cell imaging information to inform and improve our cell culture production?
Wow this a good question. With the latest technology there are many ways to measure cell health and other things that will ultimately make cell culture production better. For example Molecular Probes makes some great fluorescent dyes for just those purposes. pHrodo Indicators for example get onto cells and change colors depending on the cytosolic pH. Conjugates with pHrodo allow for visualization of ligand or antibody internalization. Also chemicals such as AlamarBlue or Live/Dead cell viability assays allow for easy detection of proliferation which is related to improvements in a cell culture process.
What molecules in media present the biggest problems with fluorescence imaging?
Many of the chemicals in media fluoresce when the right wavelength light is present. Fluorescence microscopy is often difficult since getting the correct signal:noise is dependent on the components that make up the media. Often incubations in PBS are used to reduce auto-fluorescence but there are problems with that since the nutritional content of PBS is basically nonexistent. This problem sets limits to incubation times one can use, if cell health is important. Many components of media are cyclic and conjugated and contribute to noise during fluorescence microscopy because those molecules get excited by the wavelengths used during fluorescent microscopy. Probably phenol red and flavin molecules contribute the most to background fluorescence. This is one reason many people use phenol red-free medium during these types of experiments. The problem though is that when using phenol red-free DMEM there is only a small reduction in background fluorescence compared to PBS.
What kind of system would you recommend for live-cell imaging? Would you use live-cell incubation chambers or something else. Also what are the advantages of live-cell imaging?
There are lots of good chamber systems for live imaging. If imaging is occurring for a few hours you might be able to get away without an incubation chamber but if your incubations are longer a more sophisticated system would be necessary. Here is a good website from Nikon that talks about them (www.microscopyu.com/articles/livecellimaging/culturechambers.html).
Obviously a good chamber needs to maintain incubator like conditions without condensation and the viewing port needs to be optically clean. Also a good chamber will have some ports for media perfusion as wells a heating system to maintain the appropriate growth temperature. Typically some sort of glass is used since cell culture plastic does not work well with fluorescence. Live cell imaging allows one to visualize changes in the cells over time and with appropriate tags it is possible to follow trafficking of proteins or complexes as well as visualize interactions. One of the biggest problems that make live cell imaging difficult is the background fluorescence from the cell culture medium itself. Most would opt for media without phenol-red but even this has significant background problems. This is one reason people use DPBS for this type of microscopy.
To answer some or most of these problems with auto-fluorescence and poor signal:noise, GIBCO is about to come out with a new product specifically designed to address these problems. A new product called FluoroBrite DMEM is being released that has background equivalent to DPBS and 90% less than standard phenol red-free DMEM. The nice thing about this is you can maintain optimal cell health while getting the images you want with low background.