[Paper Review] Dynamic contrast in scanning microscopic OCT

Michael Münter, Malte vom Endt+8 more|arXiv (Cornell University)|Feb 28, 2020

Optical Coherence Tomography ApplicationsEngineering25 references90 citations

TL;DR

This study demonstrates dynamic contrast in scanning frequency-domain optical coherence tomography (FD-OCT), enabling high-contrast, speckle-free imaging of cellular and subcellular structures in unstained murine tissue. By analyzing temporal intensity fluctuations in OCT signals across three frequency bands (0–0.5 Hz, 0.5–5 Hz, 5–25 Hz), the method achieves histology-like contrast in B-scan and volumetric images, revealing epithelial layers, nuclei, and muscle fibers with micron-scale resolution despite high numerical aperture objectives.

ABSTRACT

While optical coherence tomography (OCT) provides a resolution down to 1 micrometer it has difficulties to visualize cellular structures due to a lack of scattering contrast. By evaluating signal fluctuations, a significant contrast enhancement was demonstrated using time-domain full-field OCT (FF-OCT), which makes cellular and subcellular structures visible. The putative cause of the dynamic OCT signal is ATP-dependent motion of cellular structures in a sub-micrometer range, which provides histology-like contrast. Here we demonstrate dynamic contrast with a scanning frequency-domain OCT (FD-OCT). Given the inherent sectional imaging geometry, scanning FD-OCT provides depth-resolved images across tissue layers, a perspective known from histopathology, much faster and more efficiently than FF-OCT. Both, shorter acquisition times and tomographic depth-sectioning reduce the sensitivity of dynamic contrast for bulk tissue motion artifacts and simplify their correction in post-processing. The implementation of dynamic contrast makes microscopic FD-OCT a promising tool for histological analysis of unstained tissues.

Motivation & Objective

• To develop dynamic contrast imaging in scanning frequency-domain OCT (FD-OCT) for enhanced visualization of cellular and subcellular structures.
• To overcome limitations of time-domain full-field OCT (FF-OCT), such as low penetration depth, high sensitivity to axial motion, and slow volumetric acquisition.
• To enable depth-resolved, tomographic imaging with histology-like contrast comparable to H&E-stained sections.
• To assess the feasibility of dynamic contrast in scanning FD-OCT for future endoscopic and in vivo optical biopsy applications.

Proposed method

• A supercontinuum light source (550–950 nm) with a 1 µm axial resolution was used, coupled with a fiber-based Michelson interferometer for FD-OCT.
• An x/y galvanometer scanner and achromatic telescope scanned the beam to the back focal plane of a 10x/0.3 NA objective, enabling B-scan imaging.
• A-scan rates up to 248 kHz were achieved using line-scan cameras (SprintSpL4096 and OctoPlus CL), with B-scan rates of 108 Hz.
• Temporal signal fluctuations in each voxel were Fourier-transformed to extract power in three frequency bands: 0–0.5 Hz (blue), 0.5–5 Hz (green), and 5–25 Hz (red), color-coded into RGB images.
• Residual dispersion was corrected using a 5th-order polynomial, optimized via Shannon entropy to improve image quality.
• Post-processing included logarithmic scaling of color channels, contrast-limited adaptive histogram equalization, and brightness normalization to the focal peak.

Experimental results

Research questions

• RQ1 Can dynamic contrast be effectively implemented in scanning FD-OCT to enhance cellular contrast without relying on tissue fixation or staining?
• RQ2 How does scanning FD-OCT compare to FF-OCT in terms of motion artifact sensitivity and image acquisition speed for dynamic contrast imaging?
• RQ3 To what extent does high-NA objective usage affect dynamic contrast in scanning FD-OCT, particularly in terms of lateral resolution and depth-of-focus?
• RQ4 Can dynamic contrast in scanning FD-OCT reliably differentiate tissue components such as epithelium, lamina propria, and muscle fibers?
• RQ5 Is dynamic contrast robust against phase noise introduced by scanning mirrors and axial sample motion in a B-scan geometry?

Key findings

• Dynamic contrast in scanning FD-OCT successfully visualized individual epithelial cells, cell nuclei, and cytoplasmic structures in fresh murine tongue and liver tissue with micron-scale resolution.
• The method produced speckle-free B-scan and volumetric images with contrast comparable to H&E-stained histological sections, even beyond the focal plane.
• Cellular structures such as the basal layer, lamina propria, and muscle fibers were clearly identifiable, with nuclei appearing as yellow-colored regions in the dynamic contrast images.
• The technique achieved depth-resolved imaging across tissue layers, enabling tomographic visualization similar to conventional histopathology.
• Imaging speed was limited by galvo scanner, camera line rate, and relative intensity noise (RIN) of the supercontinuum source, but potential for 12 volumes per second at 600 kHz A-scan rate was demonstrated.
• Scanning mirrors did not interfere with dynamic signal extraction, and axial motion artifacts were mitigated via individual A-scan phase correction, enhancing robustness over FF-OCT.