BHA01.07 Spectrometer

Spectrometer

How could we control the amount of cells suspended in our solution? You could take a sample with a known volume, look through it in the microscope and count cells manually. This is a time consuming process and there must be a more efficient way to do so.

Three things can occur when light hits an object: it can be reflected, it can be refracted or it can be absorbed. Each atom will absorb a particular wavelength and transmit or reflect others. Each biomolecule will absorb a given set of wavelengths and transmit others. This can be considered as the footprint of the biomolecule. Imagine if we can send light with multiple wavelengths and then monitor which ones are absorbed and the amount of energy that was absorbed. This way we could extrapolate the information and then we will know the amount of cells suspended in our solution. This is the main idea behind a spectrometer.

So, we will need a source with a good spectral emission (it includes as many wavelengths as possible): Xenon lamps are known as the source with the spectral emission more similar to the sun spectra, but they are expensive. An halogen lamp has a smaller emission spectra. An LED has even smaller spectra. A laser has a unique wavelength (~630 nm for red, ~530 nm for green, ~400 nm for blue, etc). Choose your source according to your needs.

And then we need to work in the detection part. How do we read the amount and wavelength of the light that has passed through the sample? In electronics we have two devices that can do so: a photodiode and a CCD or CMOS sensor. You can find cheap CCDs or CMOS sensors in your webcam, your phone, even in your optical mouse! This is a cheap way to build your spectrometer. The easiest way will be to get a webcam, dismantle it and plug it to your computer. Then use software to read the intensity of light per area and plot the results. A photodiode is a device that changes the current that flows through it depending in the intensity of light that hits its surface. Reading the current flowing through the photodiode we could know the amount of light hitting its surface.

But how to know the wavelength of the incident light? We can use a diffraction grating to split the light into its different wavelength components. As you know, white light is comprised of the sum of all different colours (wavelengths).  Using a prism you can separate the light in all the components (the rainbow colours you observe in nature). You can use a diffraction grating instead of the prism to do the same. Diffraction gratins are found in CDs and DVDs. We choose to use a DVD grating. Get a blank DVD, if you observe the edge you will note that there are two discs pasted each other. Use a razor to split the DVD. Take the transparent part, then cut a small piece and use it to diffract the light. Each wavelength will go through a different path and you will get a nice “rainbow” pattern at the output of the diffraction grating. You can calibrate your photodiode array to indicate that a given side corresponds to the red colour and the other side to the blue colour.

The device needs to be calibrated by doing a “dark” measurement (with your light source OFF), then a “white” measurement (with your source ON) and finally a measure with your sample. By comparing this data you can get an approximation of the amount of  absorbed light and then relate it to the concentration of your sample. You could use a control solution without your sample and repeat the measurement.