Label-free intratissue activity imaging of alveolar organoids with dynamic optical coherence tomography

Abstract

An organoid is a three-dimensional (3D) in vitro cell culture emulating human organs. We applied 3D dynamic optical coherence tomography (DOCT) to visualize the intratissue and intracellular activities of human induced pluripotent stem cells (hiPSCs)-derived alveolar organoids in normal and fibrosis models. 3D DOCT data were acquired with an 840-nm spectral domain optical coherence tomography with axial and lateral resolutions of 3.8 µm (in tissue) and 4.9 µm, respectively. The DOCT images were obtained by the logarithmic-intensity-variance (LIV) algorithm, which is sensitive to the signal fluctuation magnitude. The LIV images revealed cystic structures surrounded by high-LIV borders and mesh-like structures with low LIV. The former may be alveoli with a highly dynamics epithelium, while the latter may be fibroblasts. The LIV images also demonstrated the abnormal repair of the alveolar epithelium.

Fig. 1.

The culturing and measurement protocol of hiPSC-induced alveolar organoids including normal and bleomycin models. The details of the cultivation and measurement are described in Sections 2.1 and 2.2, respectively. The day of the organoid establishment is set as Day 0. The OCT measurements were performed at Days +3, +7, +10, +16, +17, and +18. Note that this study is not longitudinal, i.e., the organoids measured at different time points were not the same. Each organoid was fixed in formalin after the OCT measurement for histological preparation.

Fig. 2.

(a) En face OCT-intensity and (b) LIV images of a normal alveolar organoid measured 3 days after the organoid establishment. The images were extracted from approximately $100\text{μ}\mathrm{m}$ below the sample surface and the FOV is $1\text{mm}\times 1\text{mm}$ . The cystic and mesh-like structures are expected to be alveoli and fibroblasts, respectively.

Fig. 3.

The LIV en face images of the Day +3 normal organoid at several depths (square panes, FOV is $3\text{mm}\times 3\text{mm}$ ). The images were extracted at a depth of every 0.07 mm, and the depth positions are indicated in the cross-sectional LIV image at the center. The alveoli with high LIV (green or green-red mixture) borders, which are possibly alveolar epithelium, and the fibroblasts are uniformly distributed in 3D. The red arc at the 0-mm depth (arrow) and the red line in the cross-sectional image (arrow) are caused by the surface reflection of the culture medium.

Fig. 4.

The en face OCT-intensity and LIV images of normal and bleomycin-model alveolar organoids at Days +3, +7, +10, +16, +17, and +18. The images were extracted approximately $\mathrm{100-}\text{μ}\mathrm{m}$ below the sample surface. The FOV is $3\text{mm}\times 3\text{mm}$ .

Fig. 5.

The en face OCT-intensity and LIV images at all time points. The samples are the same with Fig. 4 but measured with a smaller FOV of $\mathrm{1-}\text{mm}\times \mathrm{1-}\text{mm}$ . Due to the smaller pixel separation (i.e. higher pixel density) than that of Fig. 4, finer structures are visible. The images were extracted approximately $\mathrm{100-}\text{μ}\mathrm{m}$ below the sample surface. The normal model at Day +3 is the same image with Fig. 2.

Fig. 6.

The HE- and EVG-stained histological micrographs obtained from the identical samples to the OCT and LIV images of Fig. 4. Appearances similar to the corresponding OCT images were observed in all samples. In the EVG images, elastin appeared as dark blue. In the enlarged bleomycin-model organoids (the most right column), the fibroblasts appeared as dark blue in the EVG image.

Fig. 7.

Selected OCT-intensity and LIV images showing several types of alveoli. The samples are the Days +10 and +17 normal model and the Days +7, +10, +16 and +18 bleomycin model. The FOV of the en face image is $1\text{mm}\times 1\text{mm}$ . (l), (m), (o) and (p) are the cross-sections at the green and orange lines in the corresponding en face image (k) and (n). The tessellated dynamic appearance of the alveolar epithelium is denoted by the white arrows, and the ragged alveoli that encapsulate the hyper-scattering mass are indicated by yellow arrows. The en face images are a subset of Fig. 5.

Fig. 8.

(a) Counts of the tessellated and non-tessellated alveoli. Here the counts are the summations of filled and non-filled alveoli. (b) Count of all four types of alveoli. The counts in the normal and bleomycin models are plotted with dots and cross marks, respectively. The counts were obtained using en face images with a FOV of $3\text{mm}\times 3\text{mm}$ as described in Section 2.5. As shown in (a), the tessellated alveoli (red) show an increasing trend over time for the both normal and bleomycin-model organoids. In contrast, the non-tessellated alveoli (blue) show a decreasing trend over time for both models.

Fig. 9.

Area and circularity of the four types of alveoli. Each circle corresponds to each organoid, and the mean values are plotted with cross marks. Significant differences of mean and variance between the normal and bleomycin-model organoids are marked on the top and side of the plots, respectively. The dot ( $\cdot$ ), $\ast$ , and $\ast \ast$ indicate P < 0.1, 0.05, and 0.01, respectively.

Fig. 10.

The comparison of en face LIV images of a sample comprising only fibroblasts and Matrigel (a), pure Matrigel (b), and the Day +3 normal organoid (c). (c) was reprinted from Fig. 4 for reference. By comparing these images, it is evident that the mesh-like structures are fibroblasts. The FOV of en face image is $3\text{mm}\times 3\text{mm}$ .

Fig. 11.

HE stained images of the alveoli that encapsulate some masses. The sample is the bleomycin model of Day +16, and the (a) and (b) are obtained from the same sample. Cell nuclei are observed in the mass inside of the alveoli.

Fig. 12.

The SD-OCT system used in this study (a) and en face LIV images without (b, c) and with (d, e) motion correction described in Section 2.3.1. The motion artifacts are found in some sub-fields in (b) and (c) as indicated by the color boxes (blue, yellow, and green). These artifacts are not observed in the images with motion correction, see dashed color boxes (d, e). The effects of the motion correction are more readily observed in the magnified images at the right (significant points are indicated by arrowheads).