Not as much as you might think.
The camera already compensates for that in software.
Otherwise, photos would look wrong.
But generally two wavelength measurements are taken at fairly small wavelength differences, where the spectral response does not differ much. To assess the purity of a DNA/RNA sample, for example, the ratio of 260 nm absorbance to 280 nm absorbance is used. If that ratio is 180:100, the sample is pure DNA, and if it is 200:100, the sample is pure RNA. In a 20 nm range, the spectral response is the same to a fraction of a percent, and what we are looking for is a 20% difference. But even that 20% is just a rule of thumb. It is based on the absorbance of the individual nucleotides:
Guanine: 1.15
Adenine: 4.50
Cytosine: 1.51
Uracil: 4.00
Thymine: 1.47
So if your DNA strand had a lot of A and your RNA had a lot of G, you could be just plain out of luck. The rule of thumb assumes a random distribution of nucleotides.
The way you use your home-built spectrophotometer is by calibration. If you had samples of pure nucleotides, you would do the 260/280 reading, and find out what multiplier gave you 4.5 for adenine and 1.15 for guanine, and you are all set -- that automatically controls for the spectral response of your sensor.
On Mon, Feb 4, 2013 at 5:35 PM, Josiah Zayner <josiah.zayner@gmail.com> wrote:
Simon that is wavelength calibration I am talking about intensity/amplitude calibration. The fact that CCDs used in cameras do not have a linear response to light across the spectrum makes it even more difficult. See: http://www.maxmax.com/spectral_response.htm for an example of what I am talking about.--On Mon, Feb 4, 2013 at 6:31 PM, Simon Quellen Field <sfield@scitoys.com> wrote:That's what calibration is for.But for DIYBio purposes, often what you want is simply a ratio of the values at two different wavelengths, and even that can be plus or minus 10 percent and you are still happy.Identifying certain dye molecules like the chlorophylls is a matter of looking for peaks at certain wavelengths. The same goes for emission or absorption lines for chemical elements and simple compounds. For that, calibrating using mercury vapor lines is pretty easy, and the device under discussion has calibration procedures and software designed for that (the one on my web site self-calibrates automatically). As you can see from the spectrograms on that site, my design gets sub-nanometer resolution on the peaks for things like sharp emission lines. And it is similarly inexpensive, at the cost of some inconvenience. The device under discussion is more compact, and plugs nicely into your USB port for automatic analysis. Replacing the DVD with some high resolution diffraction grating (and tilting the grating to get better focus at different wavelengths) might go a long way to increase its resolution.On Mon, Feb 4, 2013 at 2:17 PM, Josiah Zayner <josiah.zayner@gmail.com> wrote:One major issue is the lack of data on the spectral response of the camera/cameras. CCDs respond to different wavelengths of light differently. Difficult to analyze any data obtained using diffraction grating because of that unless a spectral response curve is measured.
On Monday, February 4, 2013 2:52:45 PM UTC-6, Mackenzie Cowell wrote:Ah ha, calibration instructions! http://publiclaboratory.org/wiki/spectral-workbench-calibrationWell I'm interested in building one of these to play with. Will let you all know how it goes.On Mon, Feb 4, 2013 at 12:49 PM, Mac Cowell <m...@diybio.org> wrote:What do you all think about the Public Laboratory for Open Technology and Science (PLOTS) spectrophotometer design?They have developed instructions for building a basic spectrophotometer from a low-cost webcam, electrical-conduit housing, and a fragment of a DVD as a diffraction grating. Seems like the devil is in the details for something like this and they have paid a fair amount of attention to the details.Build instructions: http://publiclaboratory.org/wiki/dskWhat about calibration? I think I missed that step in their instructions...--To view this discussion on the web visit https://groups.google.com/d/msg/diybio/-/XtihuLWK_fcJ.
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