Custom optical coherence tomography/confocal scanning laser microscope system for rapid measurement of cell size

Optical coherence tomography (OCT), well developed for retinal, intravascular, and dermatological tissue-scale imaging, affords the potential for substantially increased speed of microscopic 3D imaging as compared to confocal microscopy, whose volumetric acquisition speed is limited by the need for images at multiple confocal planes to create a z-stack. Here we report the construction and quantitative characterization of a multimodal Fourier domain OCT (Fd-OCT) integrated with a confocal scanning laser microscope (CSLM) for fluorescence capability and use this inverted “OCT-CSLM” system to extract images of populations of approximately spherical 293 T cells adhering to the bottom coverslip of an imaging chamber. We demonstrate that the OCT backscatter from intracellular and cell-surface structures provides sufficient contrast to determine cell size without a fluorescent label. To extract the size of the cells with OCT, we developed an analysis method, “Adaptive Radial Boundary Estimation” (ARBE), that was first validated using synthetic OCT data sets and next used on volumetric data from the Nikon A1, the CSLM, and the OCT to estimate individual cells’ cross-sectional area, and compare the results obtained from the three different imaging systems.