Like many iGEM projects, we needed fluorescent microscopy to characterize our cells. In order to get our cells to lase, we need to shine a laser on the cells with different excitation intensities, without photobleaching the cells. A setup with a pulsed laser could be the outcome for this. However, the appropriate setup was not available in our department. Therefore, we decided to build our own microscope out of separate optical parts. By discussing our problem with photonics and optics companies, and show them our motivation to solve this difficult challenge, we were able to get all required components sponsored or borrowed, making the setup nearly cost-free. With our minimal set of available tools, we calculated and designed the optics in such a way we could image fluorescent cells while minimizing photobleaching. Our home-built optical setup did not only look impressive, but it was also crucial for characterizing the ability of our fluorescent cells to become a laser. We are using a 405nm laser and filters suitable for detection in the blue spectrum of our mCerulean (part BBa_K1890010) cells, but the setup can be easily customized to fit other projects. Each component in our setup is easily interchangeable to customize the excitation and detection capabilities for any project. We believe our specific design, as well as our approach towards solving a difficult hardware problem, can be of good use to future iGEM-teams.

In order to make a cellular laser, we cover the cell in polysilicate to create a biological ‘optical cavity’. An optical cavity is a space consisting of different refractive indices, that has the special ability to 'trap' light. Additionally, we will express fluorescent proteins in the cytosol, which will form the 'gain medium'. The photons emitted by the fluorophores will be reflected within the optical cavity, allowing them to resonate within the cell or cell culture. Once an emitted photon hits an excited fluorophore, this fluorophore will emit a photon just like the incident photon, which we call stimulated emission. This concept was discovered in 1917 by Albert Einstein (Einstein, 1917), and is the theoretical basis of our biological laser. This theory also introduces one of the key concepts required for the cells to emit laser light: in order for the light to ‘accumulate’, the majority of the fluorescent proteins should be in an excited state. Only in an excited state, an incident photon can cause stimulated emission. Therefore, if we want the cells to emit laser light, it is essential to keep the majority of the fluorescent proteins exited. We call this population inversion (Gather & Yun, 2011; Svelto & Hanna, 1976).

The lifetime of the excited state of the fluorophores is usually a couple of nanoseconds (Berezin & Achilefu, 2010), but continuously shining a laser on the cells will cause photobleaching and the fluorescence will decay within 100 µs (Jonáš et al., 2014). Due to photobleaching, we will not be able to maintain population inversion in a cell for a long time, and therefore we are not able to observe processes in the cell for a significant timespan. Therefore, we need a system where population inversion is possible without bleaching the proteins. The solution to this is using a pulsed laser with a high intensity. This way, you immediately create population inversion and you can measure whether your cell is able to lase. By allowing a long time between the pulses, there is no excitation while the protein is in its excited state (Gather & Yun, 2011), which extends the time before the bleaching occurs (Jonáš et al., 2014).

This type of excitation is often not possible in a normal fluorescence microscope, since these microscopes are not equipped with pulsing lasers. The department of Bionanoscience of the TU Delft owns a confocal microscope with pulsed lasers, which we will use for our research. However, this microscope does not entirely fit our needs, as it’s not customizable, only focuses on a part of the cell and has limited pulsing capabilities. We need to excite the entire cell with a strong pulse to cause population inversion. Therefore, we have decided to build our own microscope.

Unfortunately, the kind of setup our research requires does not allow a DIY setup with cheap materials. The required equipment is very specific, so there is only little room to improvise. Buying all this equipment was not an option, as it was way too expensive. However, we decided to see this as an opportunity instead of a problem, since building our own microscopes allowed us to collaborate and interact with one of the fastest growing fields within science: the field of photonics. Photonics companies are just now discovering their potential in the life sciences, as their classic applications were mostly material science and electrical engineering. Collaborating with these companies gave us the chance to narrow the gap between photonics or optics and the life sciences. None of these companies were familiar with iGEM and only slightly familiar with the field of synthetic biology, so it was quite a challenge for us to convince these companies that a collaboration would be of great value for both of us. Luckily, a lot of photonics companies acknowledged that the life sciences are an exciting new field for them to work in. Many companies were happy to collaborate and help us, by either sponsoring us with equipment, loaning equipment or helping us designing and building the setup.

In order to confirm whether the setup was working, we used E. coli BL21 cells that were transformed with our constitutive mCerulean BioBrick. We have previously confirmed that these cells are able to fluoresce and that they can be excited at 405nm, the wavelength of our laser. To make sure the only output we were measuring was fluorescence, and not any ‘leakage’ of light from our laser beam, we also tested cells that were not transformed with the mCerulean (part BBa_K1890010) BioBrick (OmpA-silicatein, part BBa_K1890002)). These cells are not able to fluoresce at excitation at 405nm, so if the setup is working properly we should not get a signal from these cells.

The cells were adhered to the microscope slides using 3% agarose pads and imaged at an excitation intensity of 0.5 mW. This energy is low enough to not instantly photobleach the proteins, but observe fluorescence clearly. Focusing on the cells was done manually. The results are shown in figure 7.

As we can see in figure 7B, the cells transformed with a gene for the fluorescent protein mCerulean are clearly visible. When using a strain transformed with a plasmid that is known to cause no fluorescence in the cells, in this case OmpA-silicatein, no light was observed, as shown in figure 7A. From this we can conclude that we successfully built a setup that is able to observe and measure fluorescence in a cell. There is no leakage of light of our excitation laser in the camera, since we do not observe anything when we use non-fluorescent cells. Also at a higher excitation energy (50 mW) we did not observe anything on the camera. Therefore we can conclude that our setup works as expected as it is indeed able to measure fluorescence without measuring other light sources.

More information on the results of experiments with the laser setup can be found at the Project page

Working with lasers has some serious risks. Exposure of your eyes or skin to a laser can cause burns or loss of vision. As an example: a laser pointer can already cause harm after staring at it too long, and the power of such a laser pointer is only 1 mW. Since we were working with a laser up to 100 mW, we needed to take some serious safety precautions

As a start, we all received basic laser/microscopy safety training. Furthermore, the team members that were involved in building the optical setup for the hardware component of our project also received an advanced laser safety course prior to their work in our optical lab. The laser safety of our project was supervised by Jeremie Capoulade (Head of the microscopy facility of the Department of Bionanoscience, see acknowledgements). We wore the appropriate eye protection at all times, to protect ourselves. Since our faculty moved to a new building, our laser lab at first was not classified as ML-I (hence we are not wearing lab coats in the building video). In this time, we did not test any living material in the setup. As soon as our lab got an ML-1 status, we also wore lab coats and took the right ML-1 measures.

Our lab was an optical lab, which means it was fully isolated from any light going in (to protect our measurements) or going out (for the safety of other labs). We made sure that our laser was not pointed at the door or that there were reflective components that could reflect a harmful laser beam out of the lab. Furthermore, the lab has all the requirements for laser safety, such as a warning sign and an interlock that will shut everything down when the door is opened or in case of emergency.