Angularly resolved, finely sampled elastic scattering measurements of single cells: Requirements for robust organelle size extractions

Angularly resolved elastic light scattering is an established technique for probing the average size of organelles in biological tissue and cellular ensembles. Focusing of the incident light to illuminate no more than one cell at a time restricts the minimum forward-scattering angle θ_min that can be detected. Series of simulated single-cell angular-scattering patterns have been generated to explore how size estimates vary as a function of θ_min. At a setting of θ_min = 20 deg, the size estimates hop unstably between multiple minima in the solution space as simulated noise (mimicking experimentally observed levels) is varied. As θ_min is reduced from 20 deg to 10 deg, the instability vanishes, and the variance of estimates near the correct answer also decreases. The simulations thus suggest that robust Mie theory fits to single-cell scattering at 785 nm excitation require measurements down to at least 15 deg. Notably, no such instability was observed at θ_min = 20 deg for narrow bead distributions. Accurate sizing of traditional calibration beads is, therefore, insufficient proof that an angular-scattering system is capable of robust analysis of single cells. Experimental support for the simulation results is also presented using measurements on cells fixed with formaldehyde.