WARNING: Quantum Mechanics below.
Before we even started attempting to decipher our flow cytometry data, we were faced with a peculiar problem. As we've learned, there are various gates that we need to implement in order to control for proper cell size, healthiness and, in our particular experiment, fluorescence. But, upon looking at these gates (shown to the right), we noticed some odd behavior from the GFP gate. Unlike the blue gate, the green gate seems to 'curve upwards' just awkwardly spilling over into the blue region. The BFP gate is more tame. With increasing concentrations of BFP in the cells, we created a strict boundary that *most* cells do not cross.
The wavelength spectrum range (as shown below) can be anywhere from 50-150 nm. What is important, however, is that this width can 'bleed over' and excite the detectors reserved for other wavelengths. This phenomenon does not generally occur, however, with a lower energy spectrum 'bleeding over' into a higher energy state. Very simply, this makes sense from what we know about energy levels. From the "ground state" (the lowest energy level), we can emit a high-energy electron with a blue wavelength. This high-energy electron creates a cascade of lower energy electrons (green) to jump up to another unstable state and emit yet another, lower-energy light (red in the figure below). If we emit anything greater than a blue wavelength, then we just broke conservation of energy.
What is important it is fairly easy for a higher-energy light spectrum to 'bleed over' into the lower spectrum. This is why the GFP bleed over is so interesting because we see, at large intensities of GFP expression, GFP molecules having blue-like behavior.
How can lower energy light do this? You made me suffer through this description of energy levels and now you're telling me that this may not be the explanation? Welcome to science!
So before we go farther, let me say that my hypothesis for this phenomenon is not well studied, this is my speculation based on fundamental quantum mechanics. I'm going to walk you through some of the basic principles, which will elucidate my explanation.
|Or in modern terms,|
"Plugged it into Wolfram Alpha"
Now, what we know from this 3D world is that if I throw a ball against a wall, usually it will bounce back. For those of you who played Red Butt (wall ball, butts up, rump rounders.... known by many different aliases) as a kid, you probably learned this in the most unfortunate matters. In quantum mechanics, there is a chance, a VERY small chance, but a change none the less that the ball will just go right through the wall. For those of you interseted, you can find the probability of this by calculating the DeBroglie wavelength of an object (http://hyperphysics.phy-astr.gsu.edu/hbase/quantum/debrog2.html).
|We all know the ball didn't hit the wall Johnny|
go up and pay the price
No, we aren't throwing quantum balls either.
|the black friday security guards |
stand no chance
|just plug and chug!|
In sum, the GFP 'bleed over' problem may be an artifact of a quantum particle's ability to penetrate high-energy barriers. At high enough intensities, the GFP may be able to penetrate through the blue-green energy difference and excite the flow cytometry's photodetectors. This is only possible, as we see in the GFP diagram, only at high intensity GFP for it to 'bleed over' into a higher-energy spectrum, the BFP spectrum. Notice that it keeps steadily increasing at higher intensities! Hope that you enjoyed my little intro to QM, post any questions to the comment section below.