2.1.

Induction of Colitis Model

All animal experiments were approved in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) of Peking University, and all procedures were performed in accordance with the approved guidelines of IACUC of Peking University. Male C57BL/6 mice ($n=15$), six weeks old (Beijing Vital River Laboratory Animal Technology Co., Ltd.), were kept under standard housing conditions providing water and food ad libitum. To induce colitis, mice received 3.5% weight/volume dextran sodium sulfate (DSS, 36 to 50 kDa, MP Biomedicals) in their drinking water for three days ($n=5$) and six days ($n=5$). Five mice were not treated with DSS and were used as a control group. DSS-induced model development was monitored with weight loss, diarrhea, and stool bleeding.^19–21 Prior to MRI and CLE, mice were fasted for 12 h.

2.2.

Magnetic Resonance Imaging Assessment

MRI was performed on a 1-Tesla permanent magnet small animal scanner (M3TM Aspect Imaging, Israel). To assess the anatomical changes in the mice with IBDs, we used the T1-weighted sequence (TR: 500 ms, TE: 12 ms, $195\mu \mathrm{m}$ in plane resolution, flip angle: 90 deg, acquisition matrix: $154\times 154$, number of averages: 7, slice thickness: 1 mm, and duration: 10 min and 5 s). MRI was started at day zero and repeated on day three and six after DSS induction. For MRI, mice were anesthetized with 1.5% isoflurane. Animals were kept on a water tube heating pad to keep the body temperature constant. Measurement of the diameter and thickness of the colons was performed in the ImageJ package FIJI (version 1.51).²²

2.3.

Confocal Laser Endomicroscopy Examination

Prior to CLE (CellVizio Dual Band, Mauna Kea Technologies, France) examination, the mice colon vasculature was stained via intravenous injection with $100\mu \mathrm{L}$ 2% weight/volume Evans blue (MedChemExpress). Subsequently, $100\mu \mathrm{L}$ of 0.05% weight/volume acriflavine (Sigma-Aldrich) was administered topically for mucosa cell staining.²³ Approximately 15 to 20 min after administering the dyes, CLE was performed by placing a fiber optic probe against the distal colon mucosa of each mouse. The CellVizio laser was used for confocal imaging; it generates excitation at both 488 and 660 nm and couples a dual-laser beam into the probe with 2.6-mm tip diameter, $1.4\text{-}\mu \mathrm{m}$ lateral resolution, $10\text{-}\mu \mathrm{m}$ optical sectioning, and a $60\text{-}\mu \mathrm{m}$ work distance. A CellVizio Mosaic Toolbox was used to form a bigger FOV by following the probe’s track. We quantified the vessel length, area, and diameter with the CellVizio Vessel Detection Module. The fiber optic probe cleaning procedure was followed in accordance with the manufacturer’s instructions.

2.4.

Fixation and Clearing of Colon Samples

After CLE imaging, the mice were deeply anesthetized and euthanized. Mice colons were removed aseptically, then washed with phosphate-buffered saline (PBS), and fixed with 4% buffered formalin for 24 h at 4°C in the dark. Fixed colons were dehydrated in methanol (Beijing Chemical Works, China) ranging from 25% to 100% (in PBS) for 3 h and left in 100% methanol for 24 h at 25°C. Then, colons were cleared with benzyl alcohol and benzyl benzoate (BABB at a 1:2 volume ratio) solution over 24 h at 25°C.²⁴

2.5.

Light-Sheet Fluorescence Microscopy of Mice Colons

The cleared colons were scanned with a commercial LSFM (LaVision BioTec, Germany). We combined a magnification of $2\times$ with a $2\times$ objective lens (Mv PLAPO 2VC; Olympus) covered with a 6-mm working distance dipping cap. We used a supercontinuum white-light laser (SuperK EXTREME 80 MHz VIS with wavelength from 400 to 2400 nm; NKT Photonics, Cologne, Germany) as a laser source. The filters were set as $470/40\mathrm{nm}$ excitation and $525/50\mathrm{nm}$ emission for acriflavine and $640/30\mathrm{nm}$ excitation and $690/50\mathrm{nm}$ emission for Evans blue for the detection of cell morphology and vessels in the samples. The step size was set to $5\mu \mathrm{m}$ and a total range of up to 2 mm for colon transversal scanning. The measurements were performed with 385-ms exposure times per slice, and a total imaging time of $\sim 6\min$ per colon sample. Imaris software (Bitplane, Oxford Instruments Company) was used to generate 3-D reconstructions of the tagged image file format images of the colons.

2.6.

Histological Validations

To validate CLE and LSFM findings, mice colon sections were collected and preserved in 4% buffered formalin for histologic study. The $4\text{-}\mu \mathrm{m}$ coronal sections of paraffin-embedded mouse colons were cut and stained with hematoxylin and eosin (H&E) for histological scoring.²¹

2.7.

Statistical Analysis

Data were represented as $\text{mean}\pm \text{standard deviation}$. Statistical analysis was performed in SPSS (IBM, version 23). The results of the two groups were compared using two-tail student’s $t$-tests. Correlations were analyzed using Pearson’s correlation coefficient (Pearson Product Moment Correlation) and significances were tested using two-tail student’s $t$-tests. The differences with a $p$-value $<0.05$ were considered statistically significant. Here, * denotes $p<0.05$, ** denotes $p<0.01$, and *** denotes $p<0.001$.

3.1.

In Vivo Monitoring of Luminal Changes by MRI

To monitor a transversal colon area for DSS-induced mice, MRI was performed at three and six days after DSS water feeding.²⁵ A significant increased colon lumen area was observed in DSS-induced mice, as measured by T1-weighted imaging [Figs. 2(a)–2(c)]. The cross-colon area was $1.27\pm 0.61{\mathrm{mm}}^2$ in healthy mice and $3.0\pm 0.61{\mathrm{mm}}^2$ ($p<0.01$) and $4.05\pm 0.7{\mathrm{mm}}^2$ ($p<0.05$) in mice with DSS colitis on day three and six, respectively [Fig. 2(d)].

Fig. 2

T1-weighted MRI of luminal changes: (a) the colon transversal area (white arrows) for healthy mice, (b) for three days after DSS induction, and (c) for six days after DSS induction. (d) Quantification of the colon cross-sectional area for the T1-weighted images (scale bars represent 2 mm).

3.2.

In Vivo Monitoring of Mucosal Inflammation by CLE

CLE gives high-resolution in vivo imaging of fluorescently stained cells and vessels throughout disease progression. We used the recently developed dual-channel CLE imaging system for monitoring intestinal epithelial cells and microvessels of colon mucosa.²⁶ The 488-nm laser with a fiber optic probe 0.35 mm in diameter allowed for visualization of the acriflavine-stained colonic epithelial cells and crypt architectures, as shown in the first column of Fig. 3. Healthy mice had intact colonic epithelial cells and crypts (Video 1). In DSS-colitis mice, we found severe cell damage and loss of crypt structure (Videos 2 and 3). The 660-nm laser excitation allowed for visualization of the mucosal vasculature architecture, blood flow, and vasculature leakage (the second column of Fig. 3). The merged results of the two-channel images are also shown in the third column of Fig. 3.

Fig. 3

Simultaneously dual-channel (488 and 660 nm) CLE imaging of intestinal epithelial cells (white arrows) and vessels (white arrowheads) for the control group (Video 1, MOV, 0.8 MB [URL: https://doi.org/10.1117/1.JBO.24.1.016003.1]), three days after DSS induction (Video 2, MOV, 0.8 MB [URL: https://doi.org/10.1117/1.JBO.24.1.016003.2]), and six days after DSS induction (Video 3, MOV, 0.6 MB [URL: https://doi.org/10.1117/1.JBO.24.1.016003.3]) (scale bars are $20\mu \mathrm{m}$).

To extend the FOV of CLE, the fiber probe’s trajectory was followed and the images were stitched using the mosaic technology in CellVizio [Figs. 4(a)–4(c), Video 4]. In addition, we quantitatively analyzed vessels using a vessel detection software package, which allowed for segmentation analysis of vessels, and the cumulated length, area, and diameter of the acquired images. The vessel detection analysis was $<12\mu \mathrm{m}$ in diameter for healthy mice [Fig. 4(d)], in which the mean vessel diameter was $11.1\mu \mathrm{m}$ and the total vessel length was $2550\mu \mathrm{m}$.

Fig. 4

CLE images stitching of fluorescently labeled epithelial cells and vessels: (a) intestinal epithelial cells and (b) vessels (Video 4, MOV, 0.5 MB [URL: https://doi.org/10.1117/1.JBO.24.1.016003.4]). (c) Merged dual-channel mosaic image. The vessels were detected under $12\mu \mathrm{m}$ in diameter for (d) segmentation and (e) distribution analysis using CellVizio vessel detection software (scale bars are $50\mu \mathrm{m}$).

3.3.

In-Toto 3-D Visualization of Colon Vessel Architecture and Morphology by LSFM

LSFM has $\sim 10$ times higher resolution than MRI. After MRI and CLE imaging, mice were euthanized, and the colons were removed for tissue clearing [Fig. 5(a)] by adapting the BABB protocol.²⁴ The colonic mucosa and microvasculature were assessed in 3-D LSFM [Fig. 5(b), Video 5]. LSFM datasets showed disrupted colon villus structures and severe loss of mucosal architecture in three and six days DSS-inducted mice, respectively. We found that colon thickness and length were significantly different between healthy mice ($n=5$) and mice with DSS-induced colitis ($n=5$). After six days of DSS induction, we observed a significant increase in colon thickness in DSS-colitis mice [$0.5\pm 0.05\mathrm{mm}$ versus $0.25\pm 0.05\mathrm{mm}$, $p<0.01$, Fig. 5(c)]. Colon length was $85.75\pm 3.6\mathrm{mm}$ in healthy mice and $63.7\pm 8.5\mathrm{mm}$ ($p<0.05$) in DSS-colitis mice.

Fig. 5

LSFM imaging of fluorescently labeled epithelial cells and vessels. Illustration of the mouse colon before and after clearing by BABB protocol. (a) The distal part of a cleared colon was selected for LSFM imaging. (b) Cleared LSFM images of acriflavine- and Evans blue-stained intestinal epithelial cells (left column) and vessels (middle column) (Video 5, MOV, 2.9 MB [URL: https://doi.org/10.1117/1.JBO.24.1.016003.5]). Quantification of (c) colon thickness and (d) length. Scale bars are 1 cm in (a) and $50\mu \mathrm{m}$ in (b).

3.4.

Histological Evaluation of DSS-Induced Colitis

To characterize the development of DSS-induced colitis and validate MRI, CLE, and LSFM imaging results, histological alternations of healthy and DSS-induced mice after three and six days were assessed by H&E staining of paraffin-embedded colon sections. As DSS-induced mucosal damage progresses, the increasing ulceration and inflammation with the loss of crypt structure were observed [Figs. 6(a)–6(c)]. A histological scoring system based on epithelial damage and inflammatory infiltrates was used to quantify the severity of the colitis²¹ [Fig. 6(d)].

Fig. 6

H&E staining of paraffin-embedded transversal colon sections for histological changes analysis in (a) healthy mice, (b) DSS-induced mice after three days, and (c) DSS-induced mice after six days (scale bars are $100\mu \mathrm{m}$). The black arrow represents the area of severe transmural inflammation with the loss of crypt structure. Black arrowheads indicate edematous submucosal inflammatory infiltrates. (d) Histological scores of the control group and DSS-induced groups (day three and six).

3.5.

Correlation of MRI, CLE, and LSFM

To correlate in vivo and ex vivo findings, we calculated the correlation coefficients of MRI (cross-sectional areas), LSFM (colonic thickness), and CLE (the crypt architecture, microvascular alteration and fluorescein leakage classification score)⁶ results with respect to weight loss, colon length, histological score, and the inflammation-associated activity index from healthy and DSS-induced mice (after three and six days); this correlation analysis is detailed in Table 1. The MRI results had a strong correlation with weight loss, and a significant correlation with the colitis activity index, colon length, and the histological score. The 3-D LSFM results were significantly correlated with the in vivo evaluation of weight loss and the disease activity score. In addition, it was strongly correlated with colon length and the histological score determined by postmortem evaluation. The in vivo macroscopic imaging technique, MRI, was significantly correlated with the parameters representing the development and severity of colitis, and microscopic level results, observed using the LSFM imaging method, were also significantly correlated with these parameters.

Table 1

Correlation of the mean values from MRI (transversal areas), LSFM (colonic thickness) and CLE (the crypt architecture, microvascular alteration, and fluorescein leakage classification score) with body weight loss, colon length, histological score, and the inflammation-associated active index.