Difference between revisions of "Team:Grenoble-Alpes/fluorescence module"
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<p><font color="white">As a conclusion, here are some advice you should follow if you need to create your own fluorescence sensor, to avoid painful troubles:</p><p> | <p><font color="white">As a conclusion, here are some advice you should follow if you need to create your own fluorescence sensor, to avoid painful troubles:</p><p> | ||
Revision as of 09:35, 9 October 2018
Template loop detected: Template:Grenoble-Alpes
FLUORESCENCE MODULE
In our Top10 competent bacteria, we previously inserted, thanks to a bacterial transformation process, the pSB1C3-BBa_J04450 backbone that contains a gene coding for mRFP1. Hence, when the bacteria will express this gene, they will start producing fluorescence, which is related to the presence of the pathogenic bacterium.
The fluorescence expressed by the Top10 comes from the mRFP1 protein, whose spectrum is given in figure 1.
This fluorescence is detected thanks to our home-made unit which is shown here:
Light pathway through the fluorescence sensor
Excitation light
To excite the fluorophore, it is necessary to use a light which emission spectrum is the narrower possible. Therefore, LEDs are much appreciated as their emission spectrum are usually very narrow. Moreover, they are easy to supply (they just need a resistance to protect them and keep the light intensity constant) and very small. We used 565 nm LNJ309GKGAD LEDs (Panasonic) whose spectrum is given in Figure 3.
CONCLUSION
As a conclusion, here are some advice you should follow if you need to create your own fluorescence sensor, to avoid painful troubles:
- Unless you have a photomultiplier available with the appropriate supply, make a camera your first choice before a photosensitive device, such as a photoresistance or a photodiode. The fluorescence analysis is highly limited by the fluorescence intensity. It is easier to see fluorescence than detect it. Hence, with a photosensitive device, the minimal sensitivity will not suit your application. Photosensitive devices are helpful when you try to do quantitative measurements, as they directly link the light intensity to their output, but it is meaningless if you cannot detect a low-level fluorescence. If a photosensitive sensor is mandatory for your application, you should use an optic fiber to focus the light to the sensor with no losses.
- Be careful of the material you work with. The plastic you use, for instance, might be fluorescent as well, making your results wrong. The material might also reflect the excitation light to the sensor when you expected it to be confined.
Look carefully at the characteristics of the excitation light. The brighter the better it is to boost the fluorescence intensity, obviously, but it is not enough. A high angle of vision (more than 100°) can be interesting to illuminated a large surface, but beware not to illuminate your sensor. A small angle of vision would ensure that it does not happen but the resulting light intensity would be usually lower.
For the sake of reducing the noise level, as usual, you should consider using an excitation light with a lower central wavelength than the optimal one in case the excitation spectrum of the fluorescent protein overflows on the emission spectrum, and hence taking advantage of the excitation spectrum spreading (taking a higher cutoff wavelength longpass filter works as well).