Introduction: Measuring the Invisible

As we were planning our project we considered the yellow fluorescence protein mVenus to be a straight-forward screening tool. With our first successful transformations we realised how wrong we were. Equipping an organism full of pigments and photoreceptors like C. reinhardtii with a fluorescent protein for concentration measurements is quite brave, but using it for screening seemed to us to be a manageable task. Our first plate reader results taught us otherwise. With every measurement indicating a higher or equal fluorescence for wild type algae than for the transformed, frustration started to rise. Measuring YFP in C. reinhardtii turned out to be like looking for a fluorescent needle in a colorful haystack.

Our first approach was to perform colony PCRs on every YFP-construct colonies that were picked, something we were trying to avoid. We had hoped to be able to use YFP as a fast screening tool for our successful transformations. Every clone that showed a positive band in the PCR gel was then observed under a fluorescent microscope. At this point we were able to identify our first YFP clones and that the part worked in principle. Returning to the plate reader our now confirmed YFP clones still showed smaller or equal emission values than our wt strain UVM 4 under the plate reader. One thing we did achieve: more frustration. Only after many measurements and trial and error we were able to identify the correct parameters to measure our YFP. We are aware that for many experts in the field of fluorescence measurement this kind of measurements are part of the daily routine. Nevertheless, our goal is to expose our mistakes and troubleshooting when measuring YFP in C. reinhardtii to help other young researchers and iGEM teams that might find themselves in a similar situation.

Our Mistakes: What You Can Do Better

There are many factors that play an important role when measuring fluorescence. The first important thing to take into account is the physics of the measuring device. Most fluorescence spectrometers have a 90° angle of excitation and emission length. This greatly improves the quality of the measurement by decreasing drastically the mixing of excitation and emission light. Nevertheless, there are spectrometers and other measuring devices, like plate readers, that measure the emission light in the same angle as the excitation light. This leads to the problem that you measure your own excitation light as part of the emission signal and thereby increase the input into the sensor. This can lead to false positive measurements, over excitation of the sensor and many more problems, especially if the emission and excitation wavelengths are close to one another. This was a problem we had to deal a lot with. Having access to a spectrometer or device with a 90° angle of excitation and emission is indeed of great help.

The emission and excitation peaks of mVenus are very close to one another. The excitation peak is at 515 nm and the emission peak at 528 nm (Kremers et al., 2006). This led to the problem that, due to the same angle of excitation and emission light, we could not excite at 515 nm and measure emission at 528 nm. A very relevant factor is the band width of the excitation and emission. A narrow bandwidth allows measurement were excitation and emission are close to one another. A wide bandwidth demands the use of emission and excitation wavelengths that are wider apart. We had to learn this by adjusting our measurements to the bandwidth of our plate reader.

Another important factor when measuring fluorescence in organisms is to make sure that you have a comparable cell concentration in your probes. What we hoped to achieve with our YFP was to screen for positive mutants: if fluorescence was detected, it meant that our part was successfully being expressed. Yet, we encountered problems with the cell concentrations when they varied in our assays. For example, a positive clone with a low cell density in the probe would appear to have lower fluorescence than a negative, non fluorescent clone with a high cell density. False positives would be measured and positive clones would go unnoticed. By adjusting and equaling the cell concentration of all your probes you can assure a better chance of success when measuring fluorescence emission of your probes. Unfortunately, this sometimes can require an effort that ultimately defeats the purpose of fast and efficient screening, especially when using 96-well plates.

Additionally, regarding the pigments, photo systems and light antennae in C. reinhardtii, it must be taken into account that there are fluorescence quenching effects. The emission light of YFP is absorbed by the pigments, photo systems and light antennae of the algae in the probe, therefore reducing the measured emission.