Fluorescence detection in living cells and ways to improve your image


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. Although in theory, fluorescence microscopy is simple, obtaining suitable images is difficult. Problems with cell health can occur due to long incubations in D-PBS in an attempt to reduce auto-fluorescence. Cell death due to light intensity or photo-bleaching can be problematic and need to be overcome. If you are having problems with signal:noise or cell health or obtaining the best image possible now is your chance to ask the expert.

Question 1

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.

Question 2

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 (http://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 trafficing 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.

Question 3

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.

Question 4

What would you recommend for label-free imaging and do you see genetic expression of fluorescent proteins being a challenge to imaging?

I am not sure that I understand the questions I am thinking that you are asking about something like GFP or one of the variants. To view GFP, most people use FITC filter sets and you will also need fluorescence capabilities. From your second question I don't think it is too challenging to express GFP or a similar protein, and view many interesting things depending on what the protein partner is, what is does and where is goes etc. If you are interested in long term imaging you will need an on-stage incubator. I have included a website here that will give you information of a great set-up for the work you are interested in doing. (http://www.lifetechnologies.com/us/en/home/life-science/cell-analysis/cellular-imaging/cell-imaging-systems/evos-fl-auto/evos-onstage-incubator.html)

Question 5

What do you recommend for evaluating cell viability with fluorescent imaging?

It depends on what you want to do. Newer fluorescent assays for cell viability work the same way as the older non-fluorescent counterparts. Viability assays work by taking advantage of membrane permeability loss in compromised cells. Therefore the dyes are influxed into the cell where they bind DNA. Unbound dyes do not fluoresce. An example is the HCS LIVE/DEAD Green Kit or the LIVE/DEAD CEll Imaging Kit. The HCS Mitochondrial Health Kit also indicates mitochondrial health for ever better sensitivity to cell health. The reason I like these kits are that they are very easy to use and they are reliable. Here is a link for more information. http://www.lifetechnologies.com/us/en/home/life-science/cell-analysis/cell-viability-and-regulation/cell-viability/mammalian-cell-viability.html

Question 6

What do you recommend for imaging of non-adherent cells?

You can do imaging on suspension cells. If you are using phase-contrast you won't get much since the light reflects off of the cells in suspension, but you can get images using other types of microscopy. Confocal and fluorescent imaging is possible. Often fixations are necessary. The trick is using an ultra-thin layer of cells and short working distance. If you do a search on the internet for whatever imaging you want to do and use the word "suspension" you will find a protocol.

Question 7

What methods are best for looking at intracellular events?

Hi I have to tell you there are many methods and probes for looking into intracellular events. I could not do the topic justice in a short answer. Here is a link to the Molecular Probes Handbook which will have all the answers to your questions. http://www.lifetechnologies.com/us/en/home/references/molecular-probes-the-handbook.html Have a look.

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