Effects of mismatched refractive indices in aquatic flow cytometry

Background: Forward-angle light scatter, as measured by flow cytometry, can be used to estimate the size spectra of cell assemblages from natural wate rs. The refractive index of water samples from aquatic environments can dif fer because of a variety of factors such as dissolved organic content, alde hyde preservative, sample salinity, and temperature. In flow cytometric ana lyses, mismatch between the refractive indices of the sheath fluid and the sample causes distortion of the forward-angle light scatter signal. We meas ured the effect of this mismatch on cell size measurements. Methods: We examined the error by measuring the scatter signal of a variety of particle types and sizes and changing the sheath-to-sample salinity rat io. The effects were characterized for standard microspheres, cultured phyt oplankton cells of different sizes, and natural populations from an estuari ne river. Results: We found that the distorted scatter signals resulted in an increas e in the apparent size of small cells (1-2 mum) by a factor of 4.5 times. C ells in the size range of 3-5 mum were less affected by the salinity differ ences, and cells larger than 5 mum were not affected. Chlorophyll and phyco erythrin fluorescences and 90 degrees light scatter signals were not change d by sheath and sample salinity differences. Conclusions: Care must be taken to ensure that the sheath and sample refrac tive index are matched when using forward light scatter to measure cell siz e spectra, especially in estuarine studies, where salinity can vary greatly . Of the factors considered that can change the sample refractive index, sa linity gradients in an estuary cause the largest index mismatch and, conseq uently, the largest error in scatter. (C) 2001 Wiley-Liss, Inc.