While optical coherence tomography (OCT) provides a resolution down to 1 µm, it has difficulties in visualizing 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 the site-dependent active 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), which we believe has crucial advantages. 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. Dynamic contrast makes microscopic FD-OCT a promising tool for the histological analysis of unstained tissues.

Volumetric imaging of dynamic processes with microscopic resolution holds a huge potential in biomedical research and clinical diagnosis. Using supercontinuum light sources and high numerical aperture (NA) objectives, optical coherence tomography (OCT) achieves microscopic resolution and is well suited for imaging cellular and subcellular structures of biological tissues. Currently, the imaging speed of microscopic OCT (mOCT) is limited by the line-scan rate of the spectrometer camera and ranges from 30 to 250 kHz. This is not fast enough for volumetric imaging of dynamic processes in vivo and limits endoscopic application. Using a novel CMOS camera, we demonstrate fast 3-dimensional OCT imaging with 600,000 A-scans/s at 1.8 µm axial and 1.1 µm lateral resolution. The improved speed is used for imaging of ciliary motion and particle transport in ex vivo mouse trachea. Furthermore, we demonstrate dynamic contrast OCT by evaluating the recorded volumes rather than en face planes or B-scans. High-speed volumetric mOCT will enable the correction of global tissue motion and is a prerequisite for applying dynamic contrast mOCT in vivo. With further increase in imaging speed and integration in flexible endoscopes, volumetric mOCT may be used to complement or partly replace biopsies.

In the imaging of airway tissue, optical coherence tomography (OCT) provides cross-sectional images of tissue structures, shows cilia movement and mucus secretion, but does not provide sufficient contrast to differentiate individual cells. By using fast sequences of microscopic resolution OCT (mOCT) images, OCT can use small signal fluctuations to overcome lack in contrast and speckle noise. In this way, OCT visualizes airway morphology on a cellular level and allows the tracking of the dynamic behavior of immune cells, as well as mucus transport and secretion. Here, we demonstrate that mOCT, by using temporal tissue fluctuation as contrast (dynamic mOCT), provides the possibility to study physiological and pathological tissue processes in vivo.

Purpose Microscopic optical coherence tomography (mOCT) has an imaging resolution of 1 µm in all voxel dimensions, but individual epithelial cells are difficult to resolve due to lack of scattering contrast. Adding dynamic contrast processing to mOCT (dmOCT) results in color images that enable visualization of individual cells and possibly give information on cellular function via the calculation of a motility coefficient. We propose this technique as a novel method of evaluating the ocular surface after exposure to a toxic chemical, benzalkonium chloride (BAK). Methods Ex vivo cross-section images were acquired with a custom-built, frequency-domain mOCT system. Eyes were explanted from healthy adult C57BL/6 mice and imaged every 30 minutes with five sets of dmOCT scans at each imaging time. Total epithelium and stroma thicknesses were measured from a single mOCT B-scan, and measures of color changes (hue) and the motility coefficient were acquired from dmOCT scans. Results After 30-minute exposures to 0.005% BAK, local motility decreased and total epithelium thickness increased compared to controls. For basal epithelium cells, local motility decreased after 60-minute exposures, and the hue shifted red after 90-minute exposures. Stroma thickness did not significantly swell until 150-minute exposures to BAK. Conclusions dmOCT allows us to view the behavior of the cornea epithelium under toxic stress due to BAK, revealing parallel swelling of the extracellular matrix and changes in local subcellular motion. Translational Relevance The evaluation of the cornea epithelium using dmOCT is helpful to our understanding of the toxic effects of BAK.

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