More on How the Instrument Works1. Light Transduction: the CMOS CameraComputers store all data as binary numbers (zeros and ones). How does the CMOS camera in a cellular telephone or digital camera convert light into numbers? There are several steps.
2. Software: Converting JPG Files to Intensity PlotsThe JPG format is reasonably well described in Wikipedia. The software loads ANY JPG file -- it has no knowledge what is in the image. After the user identifies the range of pixels that is to be considered a spectrum, the software reads out the Red, Green, and Blue bytes. If only a single row of pixels is selected, one simply reads out R, G, and B, then adds the 3 values to get a sum, construed as total intensity (since the values of the bytes are integers 0 to 255, the highest total intensity possible is 3*255 = 765). If multiple rows are selected, all pixels at the same offset from the blue end of the spectrum are added together. Thus, if one has a spectrum height of 9 pixels, each color runs from 0 to 255 in each pixel, so 0 to 9*255 = 0 to 2295, and total intensity can be as high as 3*2295 = 6895. The software provides options to plot the intensity from the individual color sensors or for all summed together. The abscissa may be either pixel number (no wavelength attributed to a particular offset from the blue end of the spectrum) or wavelength (calibration as given by selecting reference points in the image and presuming linear dispersion between the two reference points). 3. Software: Converting Intensity to Transmittance and AbsorbanceThe software has NO capability for stray light or background subtraction! It simply computes, at each wavelength, T = I/I0, and A = -log10 T. If the dispersion (range of wavelengths per pixel) is different for sample and reference images, the software performs linear interpolation to find an approximate intensity between measurements. In all cases "intensity" (the I or I0) is just the number computed for total intensity in step 2 above. 4. Software: Why Moving Data to a Spreadsheet May Be UsefulWhat if you want to subtract background? What if you don't want to say Itotal = Ired + Igreen + Iblue? What if you have some other manipulation you want to try? The software outputs a .CSV (comma-separated variable) file that can be read by most spreadsheet programs. To avoid making massive, uninterpretable files, it puts out only the data selected from within the JPG (a line of pixels or pixels averaged over some height), listing both pixel number and imputed wavelength. |