Spectroscopic analysis of volumetric OCT data for the automated measurement of scatterer size.

Spectroscopic optical coherence tomography enables an accurate estimation of scatterer size by computing the correlation distance (CD) function. For calibration and accuracy verification, polystyrene spheres are commonly used as size standards. However, anomalies have been observed when using the CD function to analyze spherical scatterers, which we link to multiple scattering. We have developed a robust, automated algorithm to calculate the size of hundreds of scatterers within a volumetric OCT image while accounting for the effects of multiple scattering. We measured 5.1, 7.7, and 11.3 µm polystyrene beads suspended in collagen hydrogel, agarose, and polydimethylsiloxane, which resulted in average diameters in agreement with the theoretical size to within ±λ/2, using this analysis approach. Accurately accounting for these multiple scattering effects is crucial for a robust calibration, and these measurements point the way toward analyzing the nuclear size of cells throughout a 3D tissue volume.