This post continues the previous post.
I encountered a serious dilemma in choosing the right protein. This is also a great opportunity to learn about the many properties of fluorescent proteins.
Let us start with the obvious: excitation and emission maxima and spectra.
We all know that EGFP has an excitation maximum at 488nm. That is, EGFP protein that is excited with photons at 488nm will give its maximum emission intensity at its emission maximum, 509nm.
However, we must remember that this is the maximum emission. The emission spectra is much wider, and for GFP it goes from ~470nm up to ~630nm. Figure 1 shows the EGFP excitation (dashed line) and emission (full green) spectra.
And now, what if we want to look simultaneously at two colors, EGFP and dTomato?
Figure 2 (upper panel) shows you that excitation at 488nm (the max ex. of EGFP) also excite dTomato, leading to almost 30% emission at its peak of 581nm. Therefore, if we would collect all the emitted photons, we would detect the combined excitation of EGFP and dTomato.
We therefore use filters with a narrow band of wavelength (fig. 2, bottom panel). Thus, if we use the 510/20 filter, we would detect only photons emitted at the band, in this case only from EGFP. If we use the 580/30 filter, we would detect photons coming from dTomato and EGFP.
Now what happens if we add the long-stokes shift protein LSSmKate1 to the system? Figure 3 shows LSSmKate1 is maximally excited at 463nm (red dashed), and has max. emission at 624nm (full red) [note- I drew the LSSmKate spectra, based on prior publications]. However, 460nm also excites EGFP and dTomato. The EGFP signal, at 624nm is negligible. However, dTomato gives emission at 8-9% of its maximal emission. Is this negligible? We will soon learn.
Now let’s look at mCherry. With excitation laser at 460, we have virtually no excitation of mCherry. We would therefore prefer to use mCherry in our 3-color system, instead of dTomato.
This table summarizes the excitation & emission maxima:
|FP||Max ex.||Max em.|
This table shows the relative emission of the different FPs with different excitations (the LSS data is estimated based on publications, since the BD
spectrum viewer does not include LSSmKates in its database):
|% from max emission||% from max emission|
However, excitation & emission spectra are not the only considerations. There are other parameters to consider.
Brightness, QY and EC
Different fluorescent proteins have different brightness: some are very bright, some are dim. As a matter of history, we usually consider the relative brightness, compared to EGFP (which is set to 1.00). You can see in the table below the relative brightness of the proteins discussed above.
The brightness is determined by two parameters: quantum yield (QY) and Extinction coefficient (EC).
QY is simply the ration between the number of photons absorbed to the number of photons emitted. If QY=1, then for each absorbed photon you get one emitted photon. However, we never get 100% efficiency. The protein with the highest QY that I encountered is called ZsGreen with QY=0.91.
EC (or ε) relates to the formula A = εcl in which A is the absorbance, l is the path length (in cm, usually) and c the concentration in Molar units. ε, in M-1cm-1 units, is a measurement of the capability of a certain fluorescent protein to absorb light at a certain wavelength.
The formula ε*QY gives a measure of the brightness, so for EGFP, the brightness is 55,000*0.60=33,000
For ZsGreen, the EC is only 43,000 so the relative brightness is only 1.18. This example shows that a high QY does not necessarily mean a very high brightness.
This table summarizes al the brightness data:
|FP||QY||EC(M-1 cm-1)||Brightness||Relative brightness|
Let’s go back to our dilemma: dTomato or mCherry?
We now know that with a laser excitation of 460nm, we get 100% emission from LSSmKat1 at 624nm, and only 9% emission from dTomato at 624nm.
However, dTomato is 24-fold brighter than LSSmKate1. If we look only on the QY data, at 8% emission, dTomato will produce ~6 photons for each 100 photons (100*0.69*9%), whereas LSSmKat1 will produce 8 photons (100*0.08*100%). In other words, although we get very low emission of dTomato, relative to its maximum, it is almost as high as the LSSmKate1 emission in that wavelength. Since eventually, we just “see” the photons with no knowledge of their source, the signal that we will get will be ~40% from dTomato. This will create a problem if we wish to detect changes in fluorescent intensity over time due to changes in protein localization (or other changes) because we will not be able to know if the change is due to dTomato or LssmKate1.
That is why mCherry is a better choice than dTomato for this experiment.
Another parameter to take into consideration is the protein’s photostability.
Photostability is a measure (in seconds) of how long it takes for half of the number of proteins to bleach at the maximum excitation. This matter is important for any experiment involving fluorescent proteins (or dyes) but is crucial when doing live imaging, particularly for time-lapse experiments.
LSSmKate1 is more stable than LSSmKate2 (see table below), and therefore, it might be a better choice than LSSmKate2 for my time-lapse experiment.
There are other parameters to take into account, some of them I discussed in earlier posts.
pH stability (and the effect of pH on the absorption and emission spectra).
Maturation rate – how long does it take your fluorescent protein to fold correctly and create the chromophore? If you are using your protein to measure expression rate, and the maturation time is longer than the time frame of your experiment, then you will not get the information you are looking for. Maturation rate depends on protein characteristics, as well as oxygen levels and temperature. Several fast-folding FPs were developed in recent years.
Oligomerization state – many FPs are naturally monomeric. However some FPs particularly in the orange & red range) are dimeric or tetrameric. Since in many cases the FP is genetically encoded as a fusion with a protein of interest, fusing a dimeric FP may cause your protein of interest to dimerize, through the FP, thus altering its biology. If your protein is naturally oligomeric, fusing it to a dimeric or tetrameric FP may create large protein aggregates. One solution for dimeric FPs is to create a tandem fusion proteins (i.e. two FPs are fused one to the other and to the your protein).
Generally, the letter m at the beginning of the FP name indicates that it is monomeric (e.g. mCherry, mKate), d indicates it is dimeric (dTomato) and td indicates tandem dimer (tdTomato). However, there are many FPs with no indication of their oligomeric state in their name (e.g. TurboRFP is dimeric). EGFP and its many derivatives are monomeric (except at high concentrations when they form weak dimers).
Phototoxicity – although the FPs themselves do not emit harmful radiation, the excitation light can be harmful to the cell. For instance, UV light, that is used to excite BFPs, as well as some LSS FPs and photoactivatable FPs, can cause direct DNA damage, create reactive oxygen species that will cause an oxidative stress etc… This is one reason why BFPs are the least popular FPs, particularly for live imaging.