Dynamic full-field swept-source optical coherence microscope for cellular-resolution, long-depth, and intratissue-activity imaging

TL;DR

This work presents a spatially coherent full-field optical coherence microscope (SC-FFOCM) with computational refocusing and a repetitive volume DOCT protocol to overcome the depth-resolution trade-off inherent in high-NA OCT. By flood-illuminating the sample with a plane wave and employing a phase-only refocusing filter, the method preserves total scattered energy and recovers lateral resolution across depth, enabling cellular-resolution imaging throughout thick in vitro tissues. The approach is validated on MCF-7 breast cancer spheroids, revealing structural details and intra-tissue dynamics, with LIV and OCDSl metrics mapping dynamic activity and DOX-induced changes. Overall, SC-FFOCM offers enhanced imaging depth and functional insight for thick tissue imaging, with potential for non-invasive monitoring of tissue responses in 3D culture systems.

Abstract

Optical coherence tomography (OCT) microscope (OCM) uses a high-numerical-aperture objective to achieve cellular-level lateral resolution. However, its practical imaging depth range is limited by the depth of focus (DOF). Although computational refocusing can potentially provide sharp images outside the DOF, signal reduction by the confocal effect still limits the imaging depth in practice in point-scanning OCT. In addition, standard OCT cannot visualize intra-tissue activities. To overcome these limitations, we demonstrated a spatially coherent full-field OCM (SC-FFOCM) with computational refocusing. In addition, a repetitive acquisition protocol was designed to visualize intra-tissue activities (i.e., dynamic OCT imaging). The in-focus lateral resolution is 1.4 um, and the axial resolution is 6.5 um (in air) at full-width at half-maximum intensity. Three-dimensional structure and the dynamic OCT imaging using SC-FFOCM with computational refocusing was applied to human breast adenocarcinoma spheroids (MCF-7 cell line). Volumetric dynamic imaging with cellular-level lateral resolution was demonstrated over the full depth of the spheroid.

Figures (7)

• Figure 1: (a) Schematic of the optical design. (b) Front and (c) back appearances of the spatially coherent full-field optical coherence microscopy (SC-FFOCM) system. (d) Schematic of the custom aluminum plate and shoulder bolt. (e) Schematic of synchronization of light-source wavelength sweeping and data acquisition by the camera.
• Figure 2: Schematic of the relationship between aberration and phase error in the spatial frequency spectrum of the OCT signal in (a) point-scanning OCT and (b) SC-FFOCM.
• Figure 3: Schematic of artifact removal flow using a binary filter. The signals indicated by the red arrows are assumed to be artifacts in the high-frequency domain outside of the cutoff frequency.
• Figure 4: Schematic of multi-volume refocusing. The refocusing parameters are estimated by using the first OCT volume. The same filter was applied to all 32 volumes.
• Figure 5: En face OCT intensity images of spheroids (a)-(b) without drug administration, and (c) treated with 1 µ M and (d) 10 µ M DOX without and with refocusing and refocused cross-sectional images. The depths of en face images from the top of the spheroid are described at the left side of en face images and indicated as broken lines in cross-sectional images. The yellow arrowheads indicate the boundary of the core and the periphery. The scale bar denotes 100 $\muup$m.