The use of an internal standard for semiquantitative analysis of low temperature (77°K) fluorescence of photosynthetic cells

A simple method is described which permits quantitative estimation of chlorophyll fluorescence emission intensity relative to the chlorophyll concentration in sample material at 77°K. A fluorescent standard excited by light of a similar wavelength as that of the sample material but emitting at a slightly shorter wavelength is mixed with the sample in known proportions. The fluorescence yield of the various peaks of the sample are normalized to the fluorescence yield of the internal standard. The spectra are recorded using an inexpensive attachment consisting of a Dewar holder, mounted instead of the light source of any double beam or split beam spectrophotometer. A glass rod is immersed in the sample standard mixture and then placed in the liquid nitrogen containing Dewar. Exciting light is provided by a light pipe mounted at 90° to the sample holder, connected with a tungsten-hallogen lamp provided with an appropriate filter. This method has been found to be particularly useful for the quantitation of chlorophyll fluorescence emission at 77°K of photosynthetic cells or chlorophyll containing membrane fractions. For this purpose, phycocyanin was found to be a suitable internal standard.