Bio‐optical inferences from chlorophyll a fluorescence: What kind of fluorescence is measured in flow cytometry?

A comparison is made of the in vivo Chl a fluorescence per cell measured by the flow cytometer ( F cyt ) and dark‐adapted bulk fluorescence measured in a standard field fluorometer for the marine cryptomonad Chroomonas sp. (clone Chang 2). The bulk fluorescence protocol estimated the levels of the minimum ( F 0 ) and maximum ( F max ) fluorescence yields that are exhibited depending on the redox state of the photosystem II reaction center. Both F 0 and F max are known to be functions of cell irradiance history. During the illumination of control samples at growth irradiance (40 µ mol quanta m −2 s −1 ), F 0 , F max , and F cyt (EPICS V) all increased by about the same proportion. After exposure to photoinhibiting irradiance (1,700 µ mol quanta m −2 s −1 ), F max decreased and F 0 increased. Parameters of the photosynthesis‐irradiance curve verified that photoinhibition had occurred, indicating less activity at all irradiances. In contrast to bulk fluorescence measurements, relative changes in F cyt in response to strong‐irradiance treatment were much smaller than changes in F 0 , and F max . We conclude that this is because F cyt is intermediate between F 0 and F max . Multiple regression analyses suggest that, under the flow cytometry conditions used, F cyt exhibits ∼20% enhancement above F 0 , i.e. an average of 20% of the increase from F 0 to F max . Time scales of photosystem II primary photochemistry are consistent with this amount of fluorescence enhancement occurring over the residence time of the cell in the laser beam. These results suggest caution in using oversimplified interpretations of F cyt . The enhancement effect should also be considered in other instances where fluorescence is excited by a brief saturating flash, for example, some types of in situ fluorometers.