Limitations of microscopy

Microscopes have been around for hundreds of years and the technology behind these devices has been quickly developing over the past centuries. Especially fluorescence microscopy was an essential discovery for us biologists, since we are especially interested in what processes occur inside the cell. A popular approach to image intra- and extracellular processes is to use fluorescent tags to track a molecule- or gene of interest in the cell. These fluorescent tags can be imaged under a fluorescence microscope, allowing us to trace molecules and gene expression in a cell.

This popular technique has been essential for cellular research over the past decade, and has helped us to find out several important basics of life. Even though most techniques are already very far developed, it is essential that we keep developing microscopy techniques even further. When we can image every process going on in the cell, we are able to use this for our own good. We can, for example, image the mode of action of a disease, and cure this. An example of a disease that has been studied for years but still not fully understood, is Alzheimer’s disease (Hardy & Selkoe, 2002). If we could tag all molecules in a brain cell and image them, we might find what exactly causes the disease and hopefully develop a treatment. Also synthetic biology in general benefits from a good understanding of the cell. When we can trace all enzymes involved in a certain process, for example the alcoholic fermentation in yeast, we can more easily modify genes of interest and optimize this pathway for an improved production of biofuels.

Improving microscopy using biology

The chance of a photon being observed is defined by both the chance of the photon being emitted and the photon being detected (Heintzmann & Ficz, 2006). We have thought of two possible solutions to improve the observation of a photon. The first one is increasing the chance of a photon being emitted by the sample, which in this case, are our cells. We will do this by modifying the cells in such a way they are emitting more photons for the same amount of fluorophores. This technique is based on the working principle of a laser. Therefore, we will call these cells the Biolaser.

A second approach to increase the chance of a photon hitting the detector is by directing the photons towards the detector. An easy way to do this is to apply a layer of lenses over the detector to focus the light onto the detector. A layer of tiny lenses is also called a microlens array. However, these microlens arrays are hard to fabricate and their synthesis is harmful for the environment. Therefore, we will modify bacteria in such a way they will become lenses: the Biolenses.

Synthetic biology to produce biological lasers

In order to modify E. coli in such a way that it is able to emit laser-like light, we first need to understand how a laser works. A laser has three essential components: a gain medium, an excitation source and a reflective agent. The gain medium is a medium with the ability to fluoresce. This gain medium is surrounded by a reflective agent, such as a mirror. This gain medium gets excited by the excitation source, which could be either an electric pulse or an external light source. Once this gain medium gets excited, it will emit photons. Because the gain medium is surrounded by a mirror, the photons cannot escape the gain medium but will ‘bounce’ back. When one of these photons hits an excited fluorescent molecule, something remarkable happens. This molecule will release an exact copy of the incident photon. This process is called stimulated emission. The result is that the light gets amplified each time it passes through the gain medium. Therefore, with only a limited amount of fluorescent molecules we can emit a lot more light compared to ‘conventional’ fluorescence.

In this project, we will use synthetic biology to modify E. coli in such a way that the bacterium will be able to emit laser-like light. In order to do this, we have to translate two of the main components of a laser, the gain medium and the mirrors, to biological alternatives that E. coli is able to produce. For the gain medium this is easy. We can transform E. coli with fluorescent proteins that will form the fluorescent gain medium. The biological alternative for mirrors is slightly less obvious. However, we believe to have found the solution in the enzyme silicatein. This enzyme is able to synthesize polysilicate, a biological glass. By transforming E. coli with the gene for this enzyme, we can let the cells coat itself in a layer of glass that will reflect the photons emitted by the fluorescent proteins. As an excitation source we can simply use a laser to excite the proteins. A figure of our synthetic biology approach to create a biological laser can be seen in figure 4. This biolaser will be able to emit a higher number of photons compared to a cell that is merely fluorescent. Therefore, this cell will be useful in fluorescence microscopy, since even a cell with a low number of fluorophores is hypothetically able to emit a high number of photons. Therefore, this cell could be clearly imaged. Our parts, experiments and results can be found in the Experiments section

Synthetic biology to produce biological lenses

The second approach to capture more light in fluorescence microscopy is by focusing the photons onto the detector of the microscope. We will do this with lenses, since they have the ability to focus light onto an object, such as the detector of a microscope, which is schematically shown in figure 5.

In a microscope, applying this technique would mean that each photovoltaic cell of the detector gets a lens placed on it that will direct all light into each cell of the detector. This means we need a matrix, or “array”, of lenses that are only a few micrometres small. These ‘microlens arrays’ already exist, and have been shown to be a good technique to focus more light onto photovoltaic cells, including the detector of a microscope (Jutteau, Paire, Proise, Lombez, & Guillemoles, 2015). We will also use this technique. However, we will not use the conventional, chemically produced microlenses, since they are very costly and their production is difficult and bad for the environment (Nam et al., 2013). Therefore, we aim to produce our own, biological microlenses that will be much greener and environmentally friendly compared to the conventional microlenses. Furthermore, we will also apply our biological microlenses in applications, other than microscopy, where collecting light is important. One of these applications in which we will apply our biological lenses are solar cells, more information can be found on the practices page

So how do we aim to produce these biological lenses using synthetic biology? The approach is the same as for the biological lasers: we transform E. coli with the silicatein gene. This gene allows the cell to cover itself in glass, resulting in a glass sphere of only 1-2 µm small. We will test the optical properties of this glass sphere as well as its functionality as a microlens. Since the shape of lenses is of high importance, we will also research ways to manipulate the size and shape of the cells, enabling us to produce different shapes of microlenses. Also, since our biological microlens contains a core of live bacteria, we will look into ways to sterilize the lens. This way, our product will not harm the environment in any way. Our parts, experiments and results can be found in the Experiments section