Measurement of light and pH dependence of single-cell photosynthesis by fluorescence microscopy

Measurement of algal photosynthetic performance with conventional methods r equires thousands of cells obtained by isolation and subsequent cultivation . This is a time-consuming process for many species. We describe a new meth od to study photosynthetic performance of single algal cells under various environmental conditions by a combination of modulated chlorophyll fluoresc ence, light microscopy, and sample manipulation techniques. Single cell flu orescence was measured with a modulated microfluorometer integrated in an i nverted microscope. The algal cell was sucked onto the tip of a glass micro capillary and positioned in the center of the field of view of the microsco pe by a micromanipulator. A superfusion device was used to generate a flow of experimental solution of variable composition along the alga. The light dependence of Scenedesmus obtusiusculus single-cell photosystem II (PSII) e lectron flow was measured at various pH. At a high light intensity PSII ele ctron flow was inhibited at pH 6.5 and higher, while at a low light inhibit ion occurred at pH 9.5. This is in agreement with inhibition of photosynthe sis by substrate (CO2) limitation at alkaline pH. This approach can easily be extended to study the in vivo effects of other abiotic parameters (tempe rature. nutrients, toxicants, oxygen) on the photosynthetic performance of algae.