Today I had the pleasure of doing my first FCS experiment. Actually, I sat and watched as the FCS expert in the lab analyzed my cells. The purpose of my experiment was to try to determine if a certain protein (and its mutant forms) is associated with RNA in the cytoplasm or in the nucleus of yeast cells.
First point- yeast cells are small. You will need a lot of patience.
The size (x,y) of the laser beam is ~0.3-0.4 micron. that is only 1/10 of the size of yeast cell, about half or third of the size of its nucleus. Furthermore, yeast cells have vacuole that take a considerable volume of the cell (1/4-1/2). It is therefore very difficult to set the laser at the required point in the cell (be it cytoplasm or, even more difficult – nucleus). It is even more difficult to keep it stable on that spot for the duration of data collection (2-5 minutes). Also, since these are live cells, the organelles inside the cell can move around, disrupting your measurement.
Even small movements (0.1 micron) can drastically affect the reading, especially for small cells like yeast. Therefore, the cells are immobilized on the surface of the coverslide with a lectin (Concanavalin A). The proteins on the cell surface bind to the conA coated glass, thus the cells (hopefully) do not move. However, we cannot control the movement of organelles. If we measure the protein in the nucleus, and it moves, it can affect our measurements. We can usually detect sure movements by a drastic change in the intensity readings. If it happens momentarily, one can crop out the “bad” data points. However, If it happens too much during the measurement, you’re screwed – you have to start over, or pick a different cell.
All in all, we measured a total of 24 cells (3 strains, 8 cells each), and it took us only six hours.
Second point – Bleaching
I was surprised that there was no apparent bleaching of the GFP. Bleaching is dimming of the emission light, usually due to destruction of the fluorofor. However, in this experiment we used 2-photon excitation (in short, if your fluorofor is excited at 450nm, you can shine light at a 900nm wavelength. There is a very narrow, specific, area where the photon concentration is high enough that two photons hit the fluorofor almost simultaneously (about 1 femtosecond time frame). Two photons at 900nm have the same energy as one photon at 450nm (more or less). The longer wavelength reduced photodestruction of molecules. We also used low-power laser, which contributed to the stability of the protein.
Third point – Temperature.
The FCS microscope is capable of heating the objective lens, as well as the specimen stage to a fixed and stable temperature. Since yeast normally grow at 30C, we pre-warmed the objective to 30C. the yeast sample was also heated. Actually, we did not use a simple glass slide. I used deltaT dishes, which are just small dishes with thin glass for bottom. After you immobilize the yeast, you add a small volume (1.5ml) of yeast media to keep the cells viable. However, this should also be heated. The temperature can affect not only the optics of the system, but also the behavior of the molecules at the microsecond range. Therefore, whenever we changed dishes (for the next strain), we had to wait ~20min for the temperature to stabilize.
At first, I made a mistake and used a different type of dish which cannot fit in the heating stage adaptor, and the readings were really unstable.
Fourth point – measurements.
Even if your fluorescence is low, FCS can detect it and gain very useful data. The data that is gathered is, for instance, on the Intensity of the field and Brightness of the protein. The Intensity tells you how many photons are emitted in your field for a set time differential (e.g. 50 microsecond). The Brightness is a measure of the photon emission of a single particle that moves through your field. for instance, if you have Intensity of 100, if could be due to 100 particles with Brightness of 1, or 50 particles with brightness of 2.
If you know that your protein is fused to only a single GFP, but you get twice the Brightness of a single GFP, it could mean that your protein travels as a dimer. That is very important conclusion that can be derived from these experiments.
A different kind of data is the autocorrelation which we briefly touched in the previous post. This data can tell us how fast the particles are traveling through the field of measurement. A free protein would move much faster than a protein bound to a large complex (say, mRNP). It is also possible to determine the percentage of bound/unbound proteins.
A third type of information that you can measure by FCS is concentration of your particle. Based on the Intensity, brightness and mobility, one can calculate the concentration of the particle in the field. This could be useful, for instance in comparing cytoplasmic vs. nuclear concentrations, particularly if you wish to compare mutants of your protein.
Last point –
It was exhausting but interesting. I’m am still far from being an expert, but its a start. We analyzed the data for only a few measurements, but this was just to get an general idea of the expected results. We still need to systematically analyze all the data. When I have that, I may write about it here. Show some real data.