Hello, I'm working with isolated chloroplasts from spinach leaves. I am trying to measure the oxygen production triggered by light in the presence of external electron acceptors (like Hill reagent or potassium ferrocyanide) or during CO2 fixation. However, the instrument for O2 measurement (Hansatech oxygraph) has a light integrated that is broken. What do you suggest to use as light source to illuminate chloroplasts and at which power?
Hallo Paola, chloroplasts are essentially color blind. Green light is of course considerably less efficient than red or blue light, but you can drive your chloroplasts in essence with any wavelength of visible light. However, if you want to compare your measurements with those of other people, I would have a look at the light sources that were used in their publications. Traditionally, mainly white light was used. If you would look at the older literature you would notice that people were often using water filters to filter out infrared wavelengths that may heat op your sample. The activity of your sample is sensitive to the temperature and therefore it should be stable. If you work with coupled chloroplasts electron transport activity will probably saturate at rather low light intensities. If you would use acceptors directly interacting with photosystem II or uncoupled chloroplasts saturation of activity will occur at much higher light intensities. If you can illuminate your samples by up to 1500-2000 µmol photons m-2 s-1 I think you have all bases covered.
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