A method for the accurate evaluation of the organic carbon (OC) content of phytoplankton by soft X-ray microscopy (XM) was developed and applied to the picophytoplanktonSynechococcussp., whose cells are covered by extracellular polysaccharides (EPS). Based on the X-ray absorption coefficients and gray levels of the XM images, the OC content of EPS-covered cells ofSynechococcussp. (0.39–0.47 pg/cell) was found to be 2.0–2.4 times larger than that of EPS-removed cells (0.20 pg/cell). These findings suggest that soft XM could be a useful tool for evaluating the OC content of picophytoplankton and EPS without pretreatment steps. [ABSTRACT FROM PUBLISHER]