Layers and patches of phytoplankton at sub-meter scales in the vertical dimension and kilometer scales in horizontal dimensions are common features in the coastal ocean. These heterogeneous distributions of cells are fundamental to their population dynamics and the function of pelagic ecosystems. To better understand biological processes at these small scales, methods were developed to assess phytoplankton community composition and physiological characteristics based on high-resolution, in situ optical measurements. Scanning flow cytometry of discrete samples was used to determine the effects of phytoplankton and non-algal particle abundance, size, and pigment content on the spectral shape and relative magnitude of particulate attenuation, absorption, scatter, and backscatter coefficients. The slope of particulate attenuation varied with phytoplankton size and morphology, the slope of particulate absorption and the ratio of scatter to absorption varied primarily with cellular pigment content, and the backscatter ratio varied primarily with the relative abundance of non-algal particles. Determination of particle and phytoplankton characteristics from optical measurements over small spatial and temporal scales was tested with 2 independent high-resolution data sets collected from an in situ autonomous profiling system. Comparison of these high-resolution optical data with flow cytometric sample analyses generally agreed with the previously determined relationships but suggest that complex morphology of large colonial diatoms may result in higher than expected particulate attenuation slopes. High-resolution data revealed variations in community size structure and physiology that would be difficult to visualize with discrete samples or measures of total chlorophyll concentration.