Whilst fully admitting that I am a complete dork, I want to share a paper I just found. It details a procedure for using a "fluorescent bar code" to make your flow cytometry assay more efficient. It's so simple, yet so amazing.
For those of you who don't know what flow cytometry is, a brief introduction: I got into it about three years ago and I find the technique fascinating as it combines physics, chemistry, and biology. Basically, fluorescence is a property of most substances to a certain extent. Light (photons) of a given wavelength energize electrons in an atom, allowing those electrons to briefly occupy higher-energy orbitals. Once there, the electrons are not as stable as they were and fall back to the more stable orbital, one of lower energy. The energy that was present in the higher orbital is lost and emitted as a photon of a given wavelength. Often this wavelength is different from the incoming wavelength (the one that got the whole thing started), and we call this fluorescence. In other words, the excitation (incoming) wavelength is different (usually shorter) than the emission (outgoing) wavelength.
This happens all the time. Science has identified several hundred molecules that fluoresce in very predictable ways. The most famous of which is Green Fluorescent Protein (GFP), for which the discoverers earned a Nobel Prize recently. So how do we use this to our advantage?
Well, in the case of GFP, we can insert the gene sequence into a cell, like a pluripotent stem cell or a cancer cell. As the cell then goes on to do whatever it does, the gene will be translated into GFP and the accumulated proteins will fluoresce when under light of a given wavelength. After a few hundred mitotic divisions, more and more cells will produce this protein and we can track where the daughter cells go. Check out this picture of a green mouse.
This technology has been coupled to a technology called flow cytometry to yield some amazing results. Here's a link to some flow cytometry basics from Invitrogen. In flow cytometry, a suspension of cells is injected into a column of fluid in laminar flow, i.e., a flow cell. The properties of the "flow cell" cause the cells in suspension to move, single file, through the column without mixing with the flow cell. By shining a light (a laser) at the flow cell and measuring disturbances in the light at the other side, like when a cell passes in front of the light, you can count cells. Thus, flow cytometry ("cell counting"). It gets so much cooler.
If we have fluorescent molecules present on the cells, shining a light on them will make them fluoresce and emit light of a different wavelength than the excitation light. Through the use of filters, we can separate these two wavelengths (the excitation and emission wavelengths) and count when a cell passes AND if that cell has a fluorescent molecule...all at the speed of light, literally. Commonly, we're talking about measuring fluorescence at 2,000 cells per second. Without getting into the specifics about filters, scatter, and compensation, we can measure the presence/absence of up to 20+ fluorescent molecules (all emitting at their unique wavelength) on a given cell as well as the size and shape of the cell in question all at the speed of light. How freakin' cool is that?
To blow your minds just a little more, if we put two charged plates on either side of a flow cell, we can change the path the flow to the left or right. This means that we can tell the machine, "if you see a cell emitting this specific wavelength, push it to the right, otherwise push it to the left." In other words, we can sort cells according to whether or not they have a specific fluorescent tag.
This is commonly done with immunology in separating the many subsets of cells that contribute to cellular immunity (lymphocytes, B cells, monocytes, granulocytes, etc.) We can generate antibodies that are specific to certain molecules in and on a cell, label those antibodies with a fluorescent molecule, then incubate those antibodies with a cell suspension. If we have an anti-CD4 antibody, for example, all CD4+ T-cells (helper T-cells) will be labeled with that antibody. Running them through a flow cytometer will tell us how many cells in our sample are CD4+ T-cells, or we can sort them into a tube separately from the other cells in the mix.
Anyway, the point of all of this is a paper that I just came across. Here's the link. It's by Kutzik and Nolan, 2006, in the journal, Nature Methods. Currently, most flow cytometric applications involve labeling parts of cells, keeping each sample/treatment separate. Then the samples are analyzed individually. What these intrepid researchers realized is that we can label the cells, as whole, with a fluorescent molecule. The fluorescent intensity is a function of the amount (concentration) of the molecule in the cell (lower concentrations mean a dimmer signal, higher concentration mean a brighter signal). So, by labeling each sample with a unique concentration of the fluorescent molecule can allow you to then mix all of the samples together and then stain ONE sample with whatever antibodies you want. Then you only need to analyze ONE sample on a flow cytometer because you can use that first stain to separate the individual samples after-the-fact. Sorry if this has gone way over your head. It's just the coolest thing I've seen in a while. So dorky, I know, but that's what happens.
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