Difference between revisions of "Team:TU Delft/Project"
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| − | + | <p>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 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.</p> | |
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Revision as of 09:18, 17 October 2016
Hardware
Overview
Introduction
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 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.
The basis of our custom setup
In order to make a cellular laser, we cover the cell in polysilicate or a polymer of metal oxides to create a biological ‘optical cavity’, a cavity consisting of different refractive indices. Additionally, we will express fluorescent proteins in the cytosol. The photons emitted by the fluorophores will be either scattered or 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 1916 by Albert Einstein (Einstein, 1917), and is the theoretical basis of our biological laser. This 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).
Population inversion is not easy to maintain within a medium. 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). This is too short if you want to observe processes inside a cell. Therefore, we need a system where population inversion is possible without bleaching the proteins. The solution to this is using a pulsed laser which is set up in such a way that the laser pulses match the relaxation time of the protein. This way, each time the fluorophore relaxes, it is immediately excited again, but there is no excitation as long as 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. Therefore, we have decided to build our own microscope.
Bridging the gap between synthetic biology & photonics
Unfortunately, the kind of setup our research requires does not allow a cheap, DIY setup that is often seen within iGEM. The required equipment is very specific, so there is only little room to improvise, and expensive, so buying all equipment was not an option. However, a new and exciting opportunity arose, since building this 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 they are usually occupied with 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 and were happy to collaborate an help us in building the setup, by either sponsoring us with equipment, loaning equipment or helping us designing and building the setup.
Building the microscope
The microscopy setup
Our homebuilt optical setup, the microscope, should be able to excite the laser cells using a pulsed excitation laser, and should be able to measure both an image, from which we can deduce intensity, and a spectrum of the cell, since the biolasers are expected to have improved spectral resolution. Furthermore, we wanted to keep the setup as minimal as possible, due to both the time pressure and the reproducibility of the setup. A schematic picture of the design of the setup is shown in figure 3. The setup is designed in such a way that the light of the excitation laser is filtered out after it excites the sample, therefore we are sure that the only output signal we measure comes from fluorescent cells. Furthermore, the pulsed laser minimizes photobleaching. The beamsplitter in the end enables us to measure one signal twice at the same time.
Building such a setup is not as easy as it seems. Each component should be perfectly aligned in the path of the laser beam in order for the light to be guided correctly. If a mirror is only 1° off, the path of the light will be distorted. Each misalignment amplifies this effect. Therefore, all components need to be in line with each other, with is a precise and tedious work. All parts are mounted on an optical table, which is a shock-free table that allows parts to be screwed into it. The first building session cost us 9 hours, but we summarized the building with a time lapse video of only 1.5 minute!
A more detailed view of the setup:
Because the setup consists of separate parts that are essential for fluorescence microscopy, it is very versatile. In our case, the setup is designed in such a way that it can excite and measure within the blue range of the spectrum. However, since every part of the setup is very easily interchangeable the setup can super-easily be modified to any requirement. When another iGEM team requires a custom fluorescence microscope, for example one that can measure in the red part of the spectrum, changing three components (the dichroic mirror, the filter and the laser) is all it takes to do so. Because the setup is so minimal and flexible, it can be easily adjusted to any project.
Our setup consists of the following parts, hover over the parts to find out more:
The laser cells will be placed on a stage using an ordinary microscope slide. We will use a very dilute sample of our cell culture to obtain a single-cell approach.
We will use a 50x objective of Nikon. This objective will both focus the light beam of the excitation laser on the cells, and it will magnify the laser light coming from our biolaser cells.
Since the cells have to be in a horizontal plane, we have to direct the light from the excitation laser in a vertical direction. We use a broadband dielectric mirror that reflects over 99% of the incoming light with an angle of incidence of 45°, so almost all of the photons of the excitation laser are directed towards the cells and all light emitted by the cells are directed into the setup.
For the optical setup we use a OBIS LX 405nm laser, kindly provided by Coherent. The laser is suitable for exciting the fluorescent protein mCerulean, which is one of the four fluorophores we use. This protein emits the shortest wavelength of the four fluorophores we use and is therefore more suitable for lasing. If the wavelength is close to the size of the cell, it will resonate less.
The BNC 745-4C pulse generator, which was provided by Laser2000, will allow our laser to pulse. When the laser is attached to a continuous power source, the laser will emit a continuous beam. When one attaches a pulse generator, one is able to modulate the output beam of the laser. By varying the input power between 0 and 5V we can change the output power of the laser, and changing the power inlet from continuous to short pulses will cause the laser to pulse. The pulses will only be a few nanoseconds.
The dichroic mirror is used to filter out any residual light coming from the excitation laser, so we don’t measure this as output. The dichroic mirror is a 425 nm longpass mirror, which means that is fully reflects light of all wavelengths below 425 nm, including our excitation laser, which is 405 nm. The light emitted by the fluorophores will have an emission at 475 nm, so this light will pass through the mirror. This way, we only measure the light coming from our cells, but not from the excitation laser.
To make sure that the signal we are measuring is only originating from our laser cells, and is not light of the excitation laser that was not filtered out by the dichroic mirror, we use a filter. This filter only transmits light of certain wavelengths. We use a filter that reflects all light at 405 nm, which is the wavelength of the laser.
To determine if a cell is producing laser light, we would like to measure both the intensity and the spectrum of the emitted light, while also making a picture of our cells. These measurements should be done at the same time, so we are sure that the cell has all the desired properties at once. However, we do not own a device that can do these measurements at the same time. Therefore we use a beamsplitter, that will split the emission light of the cell in a 50/50 ratio, and will direct one halve to the CCD camera and the other halve to the spectrometer, so we are able to measure the output from one cell with two devices at the same time.
In order to get an image of the cells we need a CCD camera. A CCD (Charge Coupled Device) camera has a sensitive charged chip. When a part of this chip is exposed to light, an amount of electrons gathers at this place. The ‘pattern’ of electrons on the chip is then translated to a picture. The image can also be used to determine the intensity of the emitted light. The CCD camera we use was sponsored to us by Deltapix.
The main difference between a fluorescent cell and a laser cell is the spectra the cells produce. A laser has a smaller spectral range than fluorescent light. To determine if our cells also expose this behaviour, we include a spectrometer in our setup. The spectrometer was donated to us by Thorlabs.
The finished setup looks as follows:
We have successfully imaged fluorescent cells with our setup, more information on the results of experiments with the laser setup can be found at the Project page
References
- Berezin, M. Y., & Achilefu, S. (2010). Fluorescence lifetime measurements and biological imaging. Chemical reviews, 110(5), 2641-2684.
- Einstein, A. (1917). Zur quantentheorie der strahlung. Physikalische Zeitschrift, 18.
- Gather, Malte C., and Seok Hyun Yun. "Single-cell biological lasers." Nature Photonics 5.7 (2011): 406-410.
- Jonáš, Alexandr, et al. "In vitro and in vivo biolasing of fluorescent proteins suspended in liquid microdroplet cavities." Lab on a Chip 14.16 (2014): 3093-3100.
- Svelto, Orazio. Principles of lasers. Ed. David C. Hanna. London, New York, Rheine: Heyden, 1976.
