Last week I did an experiment with a fluorescent dye in solution, just to get a feel of the different parameters that affect the reading. Here I summarize the main points that one should be aware of when performing FCS experiments:
Sampling time is the time interval (∆t) in which you collect photons. The minimum of our photon detector is 25ns. However, short time intervals are not recommended due to a phenomenon called “after pulse”. I do not know the physics behind it. For a biologist like me, it is sufficient to say that there is a Gaussian-distributed probability that after each “real” photon that is detected, a “fake” photon can also be detected. The shorter the sampling time, the higher the probability of after pulse affecting your counts. In my experiment, I changed sampling time from 1µs up to 100µs. At 1-5µs I saw an afterpulse effect.
For longer sampling time I saw a different phenomenon – undersampling. Undersampling means that the measured particles move in and out of the FCS volume in a shorter time than the sampling time. For instance, the dye I used passes through the volume in 40µs (this is the parameter of diffusion time Td), so any sampling time over 40µs will be undersampling. But why does it affect our result?
The FCS methods measures fluctuations in fluorescence of the particles. That is – different fluorescent intensity when particles entering & leaving the FCS volume. If the particles enter and exit the volume in a time frame that is shorter than the sampling time, then we will just get a sum of the total photon emission of the particles, and no fluctuation. Therefore, if you know the time it takes your particle to cross the FCS volume, you should use a shorter sampling time (preferably at least half of that). In my experiment, the dye, in water solution, passes the FCS volume within 40µs. Therefore, any sampling time above 40µs will be undersampling.
Undersampling can be calculated and corrected by using a specific algorithm.
Some examples of diffusion times:
- Alexa488 dye in water solution: 40µs
- GFP in water solution: 200µs
- Free GFP in living cells (cytoplasm): 600µs
- Free GFP in living cells (nucleus): 2ms
- mRNP particle in living cells: 80ms
This is a tricky issue, since laser power depends on the wavelength of the laser and the effect depends also on the fluorophore. However, some important issues:
The laser can melt your mirrors if the power is too high. For instance, in our instrument, going over 50mW can seriously damage the mirrors.
In my experiment I tried a range for power, from 1.5mW to 30mW. In general, the more power you use, the higher the intensity of the emission (which makes sense). This is optimally described by an equation of y=aX2. If your experimental setting is good, your results should give a plot that fits as close to power of 2 as possible. Here’s my result:
At low power you get a problem of signal to noise ratio, i.e. the difference from the background reading. For that reason, I discarded the 1.5mW result from the graph above. To overcome this problem, one should take longer measurements in order to improve the statistical analysis (for instance, I collected samples at 20µs for 60sec, and got rubbish – see graph below. Had I collected samples for 10 min I would improve the statistics and might get a better result).
As we go up in power we get another effect – bleaching. Bleaching means destruction of the fluorophore so that it does not emit photons upon excitation. Bleaching depend on laser power – the higher the power, you get more bleaching. Bleaching also depend on time of exposure to the laser light, and the stability of the fluorophore (for instance GFP is much more stable than RFP). You can see at the intensity vs. power graph above that the 30mW point does not fit well to the trend-line. This is due to bleaching. If we remove the result of 30mW, the equation “improves” to y = 0.0125x2.0192.
“Up” distance (Z) from the glass-water interface
The distance Z is the distance above the glass-water interface of your sample. For best results, it is recommended that you set Z to the focal point of your laser (see image).
The distance not only affects the intensity (the further you go from that point, the less brightness, and hence less intensity, you get), but also the volume of the measurement. For shorter distances, the volume may be only partially in water, and partially the glass. For longer distances, the laser is not focused and the volume increases – hence there is an increase in the number of particles. However, Td calculation (from the autocorrelation curve) is only slightly affected by changing the Z.
When looking at fluorophore in solution, one should note that the dye may adhere to the glass, so a short distance may cause profound artifacts. When doing live cells, other parameters come into play, such as internal organs that can obscure fluorescence, membranes that limit your volume, or causes “bouncing back” of the particle into the volume as soon as it exits. Therefore, X,Y & Z should be empirically determined for best result.
The focal point of the laser can be changed by altering the distances of the lenses of the beam expander. This too should be empirically determined for each wavelength used.
Fluorophore concentration also affects the quality of the FCS. To little particles and you will have a lousy signal-noise ratio. Too high a concentration and you get a problem of “dead time”. Dead time is the time needed after a photoncounting detector fires for the detector to recover so that it is ready to register a new photon. This recovery time may be due to the detector, the processing electronics, or some combination of the two. In our lab it is approx. 10ns.
Consequently, if you have a high concentration of fluorophore, you get a high intensity – i.e. lots of photons. Due to the dead time, some of these photons may not register, leading to under-estimation of the intensity, the number of particles and further calculations.
There are other parameters that can affect the FCS measurement, such as temperature, quality of the optics, the properties of the fluorophore itself (e.g. size, brightness, stability), the solution in which it resides (water, cell cytoplasm, cell nucleus) and more.