Chronology of the prototypes
Here is a chronology of our different prototypes :
Prototype 1 (2013)
The principe used is measuring the fluorescence at an angle of 90° from the light source.
The light from the LED passes through a lense to be focused and then through a filter, to only let the excitation wavelength pass through. That filtered light will excite the sample, which will emit at another wavelength and will then be detected. In general, photodiodes or photomultiplier are used as detectors, but they are quite expensive. That is why a digital camera is used as detector. Indeed, all camera contains an RGB filter, which can be used to detect only the range of wavelength of interest. A LED that emits at the needed range of excitation wavelength is used , to eliminate the presence of a filter.
For this device : two paperboard boxes sticked together with two holes for the camera and the LED are used. The box is closed so no external light would disturb the measurement. Inside, there is a horizontal place for the sample-holder and ouside we used an Arduino as the LED current source.
After testing, the device needs some improvements. For example, a lot of the components are outside of the box (like the LED and the camera), which makes it very difficult to take a fixed picture, because everything moves a little bit at each measure. The sample is positionned horizontally, which we discovered, is not the best orientation, because of light scattering, but also because we have to open the box for each sample. This is another factor that disturbs the system by making it move. Then we need a computer for the alimentation of the Arduino (that only acts as a battery).
So the improvements made :
- The addition of a battery to replace the Arduino and a switch, to be able to turn the LED on and off individually
- The fixation of the camera inside the device to obtain more precise pictures
- Putting the sample in a vertical position to allow an easiest change between different samples
Prototype 2 (2013)
To quantify the fluorescence of a sample, it is enough to excite it with the specific absorption wavelength in a dark room, filter the light emitted by the excited sample and then quantify it.
The idea is to use a LED generating light around 488 [nm] which is the eGFP absorption wavelength. The excited eGFP then emits light around 509 [nm]. To assure that the only light which is then quantified is the eGFP one, a filter which stops the 488 [nm] wavelength but let the 509 [nm] wavelength pass is inserted between the sample and the light quantifier.
This principle is shown in this scheme : The device works with an excitatory LED which light at 488[nm] excits the sample's eGFP, which light is filtered and quantified with the photoresistance
A lens could be necessary if the light is not enough intense to be quantified by the light quantifier. It only serves to concentrate the light in one point and so on the light quantifier device.
Another way to do it is to use two filters. One for the light filtration and another dichroic filter. As in the following figure, the dichroic is able to reflects certain wavelength and let pass others. So the idea is to reflects at 45° the excitation wavelength and then let the emitted light pass, filter a second time and then quantify the light.
The light quantifier could be a simple photo-resistance. The system we used was very simple: a photo-resistance was used as the R1 place of a voltage divider so that we can measure the voltage Vout through the arduino's microcontroller’s analogic pin. The photo-resistance, as its name indicates, is a simple electric resistance which resistance change proportionally to the light it receives. More the light increases, more the resistance decreases and more Vout tends to equal Vin. Inversely, more the light decreases, more the resistance increases and more Vout tends to be null.
Representation of the voltage divider connections and the calculation of Vout : 400px