1. Introduction

The oral cavity is covered by a mucous membrane, oral mucosa, lining the inside of the mouth. The oral mucosa has multiple functions including protection of the underlying tissues from external stimuli such as microbiological pathogens, secretion of essential substances such as saliva, and a sensory function for the perception of temperature, touch, and taste [1,2]. Diverse oral mucosal disorders can be generated by trauma, irritation, bacterial infection, malnutrition or systemic diseases [3–5]. To investigate the underlying cellular mechanisms in the pathophysiology of oral mucosal diseases, most previous studies have been conducted with histological observation of ex vivo tissue dissected from preclinical animal models [6–8]. However, this approach has a limitation in investigating dynamically changing cellular processes with the diseases progression because the obtainable information is limited to the single specific time point of tissue harvesting.

During the last decade, high-resolution confocal and multiphoton microscopy has been actively utilized for direct in vivo visualization of dynamic pathophysiological cellular processes in live animal models [9–19]. Yet, it has been mostly limited to the superficial tissues such as the skin and eye [10,11], or surgically exposable internal tissues such as the liver, spleen, and kidney [12–14]. To perform a repetitive longitudinal in vivo imaging without repeating an invasive surgical procedure, chronic implant imaging window techniques such as the dorsal skinfold chamber [20], cranial imaging window [21,22], abdominal imaging window [23–25] have been developed. Notably, intravital cellular-level visualization of the tongue was demonstrated by pulling the tongue out of the mouth and mechanically fixing it [26,27]. However, for the oral mucosa inside the oral cavity, in vivo cellular-level microscopic imaging has not been achieved due to technical difficulties. The opening size of the mouth of a small animal model is too small to enable proper positioning of a common high numerical aperture (NA) objective lens with a diameter of several tens of millimeter and a short working distance of around 1 millimeter. Furthermore, neither a surgical procedure to properly expose the oral mucosa nor an imaging window technique through the thick muscle and connective tissues in the cheek has been developed. Repetitive cellular-level longitudinal intravital imaging of the oral mucosa in the natural in vivo environment inside a live small animal model remains to be achieved.

Endomicroscopy techniques have been actively developed as a minimally invasive visualization tool for the internal organs of small animal models. Confocal endomicroscopy using a resonant single-mode fiber scanner achieved high-resolution visualization of brain tumors in rat and mouse models [28]. A spectrally encoded confocal endomicroscopy capsule [29] and multi-photon microscopy [30] using a customized endoscopic probe successfully obtained in vivo cellular level images from swine esophagus and mouse small intestine/colon without exogenous labeling. A commercial rigid endoscope modified for fluorescence detection was applied to image colorectal cancer in a mouse model in vivo [31]. Using side-view confocal endomicroscopy based on a miniature gradient refractive index (GRIN) lens and micro-prism, wide-area repetitive cellular level imaging of colorectal cancer development in a mouse model was demonstrated [32]. Additionally, a needle-shaped side-view confocal endomicroscope obtained depth-wise visualization of a growing tumor of breast cancer in a mouse model [33].

Herein, we successfully demonstrated longitudinal cellular-level in vivo visualization of the oral mucosa in a live mouse in a non-invasive manner using a custom designed rotatory side-view confocal endomicroscope. Diverse type of cells including various immune cells, pericytes, nerve cells, and lymphatic and vascular endothelial cells in the buccal mucosa were successfully imaged. With non-invasive rotational and sliding movements of the side-view endomicroscope while gently touching the surface of the buccal mucosa, multi-color wide-area cellular-level visualization of the oral mucosa was achieved. Notably, by using transgenic mice expressing a photoconvertible protein, Kaede, the same area in the buccal mucosa could be repeatedly imaged up to 10 days after photoconversion. Finally, we performed longitudinal intravital visualization of the buccal mucosa in an oral contact allergy model mouse induced by 1-Fluoro-2,4-dinitrobenzene (DNFB) [8]. It revealed highly dynamic spatiotemporal changes of immune cells in response to allergic challenge such as acute infiltration of colony stimulating factor 1 receptor (CSF1R) or lysozyme M (LysM) expressing immune cells within 12 hours and a large cellular cluster formation at day 1, which was followed by dissociation of the cluster and redistribution of the CSF1R+ or LysM+ cells at days 2-3, and then recovery to the normal baseline at day 7.

2. Materials and methods

2.1 Animal

C57BL/6N mice were purchased from Orient Bio (Seongnam, Korea). CD11c-YFP transgenic mice were kindly provided by Dr. Choi at Hanyang University. LysM-GFP mice were kindly provided by Dr. Han at Yonsei University. Prox1-GFP mice were purchased (Stock no. 03106, Mutant Mouse Regional Resource Centers) and self-produced in B6 background by mating with C57BL/6N. NG2-DsRed (Stock no. 008241), CSF1R-GFP (Stock no. 018549), CX3CR1-GFP (Stock no. 005582), Thy1-YFP-16 (Stock no. 003709) mice were purchased from Jackson Laboratory (Bar Harbor, USA). Kaede mice were generously provided by Dr. Tomura and Dr. Miwa at Kyoto University. Mice (total, n = 20; C57BL/6N, n = 2; CD11c-YFP, n = 1; CX3CR1-GFP, n = 1; CSF1R-GFP, n = 5; Prox1-GFP, n = 1; Thy1-YFP-16, n = 1; NG2-DsRed, n = 2; LysM-GFP, n = 4; Prox1-GFP and NG2-DsRed double transgenic mouse, n = 1; CD11c-YFP and NG2-DsRed double transgenic mouse, n = 1; Kaede, n = 1) used in this study were maintained in a specific pathogen-free facility of KAIST Laboratory Animal Resource Center. For experiments, 8–16 weeks old male mice (20∼30 g) were used. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of KAIST (Protocol no. KA2018-64), and all procedures were performed under anesthesia

2.2 Side-view rotatory GRIN endomicroscope integrated with custom-built intravital confocal microscope system

In this work, the previously reported side-view endomicroscopy system was utilized [34]. A custom-designed GRIN-lens-based side-view endomicroscope fabricated by GRINTECH, Germany, was used. The side-view endomicroscope was composed of a high NA coupling lens (NA 0.5, IFRL-200-023-50-NC), a relay lens with 1 pitch (NA 0.1, IFRL-200-100-11-NC) and an imaging lens (NA 0.5, IFRL-200-cust-50-NC) attached with an aluminum coated 90° micro prism with base size of 1.3 mm to reflect laser beam for side-viewing. With stainless-steel tube packaging, the diameter and length of the side-view endoscope were 2.2 mm and 60 mm, respectively. To reduce the surface gap between prism and stainless-steel tube, cover slip (thickness of 0.17 mm) was attached to the distal surface of prism with topical application of UV-curing optical adhesive epoxy (NOA63, Norland Products). Additionally, to avoid potential damage to the imaging tissue, the entire distal end of the probe was covered by the optical epoxy to have a round shape. The side-view endomicroscope was fixed to a custom-designed holder previously described [34], which was capable of rotating and XY translation to achieve a 360° side-view imaging and precise positioning, respectively. In the custom-built confocal microscope depicted in Fig. 1, four laser modules with wavelengths at 405 nm (OBIS, Coherent), 488 nm (MLD, Cobolt), 561 nm (Jive, Cobolt), and 640 nm (MLD, Cobolt) were used as excitation light sources. Laser beams were combined by dichroic beam splitters (DBS1, FF593-Di03; DBS2, FF520-Di02; DBS3, Di01-R405; DBS4, Di01-R405/488/561/635, Semrock) and delivered to 2-dimensional raster-pattern scanner comprised by a polygonal mirror scanner (MC-5, aluminum coated, Lincoln Laser) and a galvanometer mirror scanner (6230H, Cambridge Technology). Imaging focal plane was formed at the distal surface of the side-view endomicroscope, which could be adjusted in the range of 250 µm by changing the distance between the proximal end of the side-view endomicroscope and the coupling objective lens (LUCPlanFLN 40X, NA 0.6, Olympus) providing field of view (FOV) of 309 × 309 µm. The fluorescence signals captured by the side-view endomicroscope were delivered to photomultiplier tubes (PMT; R9110, Hamamatsu) through dichroic beam splitters (DBS5, FF484-FDi01; DBS6, FF560-Di01; DBS7, FF649-Di01, Semrock) and band-pass filters (BPF1, FF01-442/46; BPF2, FF02-525/50; BPF3, FF01-600/37; BPF4, FF01-685/40, Semrock). Electronic signals from the PMTs were acquired by a 4-channel frame grabber (Solios, Matrox). Video-rate images acquired at frame rate of 15 Hz with frames size of 1024 × 1024 pixels. Images stored in real-time by a custom-written software based on Matrox Imaging Library (MIL9, Matrox).

 

Fig. 1. Side-view endomicroscopy for intravital oral mucosa imaging. (a) Schematic illustration of custom-built confocal microscopy system integrated with the GRIN lens-based side-view endomicroscope (DBS, dichroic beam splitter; BPF, band pass filter; M, mirror; PMT, photomultiplier tube; Obj, objective lens). (b) Photograph of the side-view endomicroscope mounted in the holder and inserted into the mouth of mouse for the intravital oral mucosa imaging.

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2.3 Intravital oral mucosa imaging

Mice were anesthetized with intraperitoneal injection of mixture of Zoletil (20mg/kg) and Xylazine (10mg/kg). Thermal probe of the homeothermic system (RightTemp, Kent Scientific) was inserted into the rectum, and a feedback-regulated heating pad was utilized to maintain mouse body temperature at 37.0 °C. For intravital buccal mucosa imaging, the side-view endomicroscope was carefully inserted into the mouth of mouse positioned on XYZ translational stage (Fig. 1(b)). To visualize the blood vessels in the buccal mucosa, DyLight 649 conjugated tomato lectin (Cat no. DL-1178, Vector Laboratories) or anti-CD31 antibody (Cat no. 553708, BD Biosciences) conjugated Alexa Fluor 647 (Cat no. A20006, Invitrogen) was intravenously injected via tail vein at 3 hours before the imaging. To visualize the circulating red blood cells (RBCs) in the blood vessel in buccal mucosa, RBCs isolated from the blood collected from a donor C57BL/6N mouse were fluorescently labeled with DiI (Cat no. V-22885, Invitrogen) and intravenously injected into recipient C57BL/6N mouse. The Kaede mouse was utilized for the repetitive visualization of the same microscopic site of the buccal mucosa for up to 10 days. A small area of the buccal mucosa of the Kaede mouse was photoconverted by a 405 nm laser illumination (180 µW, 60 seconds) during the side-view endomicroscopic imaging at day 0. The photoconversion from naïve green fluorescence to red fluorescence was confirmed in real-time. The photoconverted area was clearly distinguishable from the neighboring non-photoconverted area, enabling the repetitive longitudinal imaging.

2.4 Oral contact allergy induced mouse model

DNFB (Cat no. D1529, Sigma) dissolved in solution of mixed with 4:1 vol/vol ratio of acetone (Cat no. 67-64-1, OCI Company) and olive oil (Cat no. O1514, Sigma) was prepared and used as allergen. Under anesthesia, dorsal skin of the mice (CSF1R-GFP, n = 4; LysM-GFP, n = 3) was shaved by a hair clippers and hair removal cream. Then two times of sensitization were conducted at day −5 and day −4 by applying 20 µl of 0.5% DNFB solution to the shaved dorsal skin. And at day 0, 10 µl of 0.2% DNFB solution applied to buccal mucosa of the mice to induce oral contact allergy [8]. For statistical analysis, at least 10 sites in the buccal mucosa were imaged at day −5, day −3, 12h, day 1, day 3, and day 7.

2.5 Image processing and Statistical analysis

The single FOV images acquired for wide-area imaging were manually stitched to create a wide-area mosaic image by using the ‘Photomerge’ function of the Adobe Photoshop (Adobe Systems). Statistical analyses were conducted by using Prism software (GraphPad). All data were expressed as mean ± S.D. Quantification data were statistically analyzed by paired two-tailed t-test for comparing the changes between mice at different time points. Statistical significance was set at p-value less than 0.05.

3. Results

3.1 In vivo cellular-level visualization of buccal mucosa by side-view confocal endomicroscopy

A previously reported rotatory side-view confocal endomicroscopy system [34] was utilized for non-invasive cellular-level imaging of the buccal mucosa of an anesthetized mouse (Figs. 1(a)-(b)). The custom-designed holder was capable of 360° rotation and axial translation of the side-view endomicroscope. The side-view endomicroscope with diameter of 2.2 mm and length of 60 mm was smoothly inserted into the oral cavity through the mouth of the anesthetized mouse. An imaging focal plane was formed at the distal surface of the side-view endomicroscope gently touching the surface of the buccal mucosa. Additionally, the imaging focal plane could be adjusted in the range of 250 µm by changing the distance between the proximal end of the side-view endomicroscope and the coupling objective lens with the translator of the holder, which enabled clear imaging of individual cells inside the buccal mucosa. Using the imaging system, we successfully obtained in vivo images of individual fluorescent cells expressing fluorescence protein in the buccal mucosa of anesthetized transgenic mice (Fig. 2(a)-(f)). Mononuclear phagocytes (CD11c-YFP [35], CX3CR1-GFP [36], CSF1R-GFP [37]), lymphatic vessels (Prox1-GFP [38]), nerve cells (Thy1-YFP [39]), and perivascular cells (NG2-DsRed [40]) in the buccal mucosal epithelial and lamina propria layers were clearly visualized. Blood vessels were simultaneously imaged by fluorescently labeling vascular endothelial cells with intravenously injected tomato lectin. Notably, the video-rate real-time imaging capability of the custom-built confocal microscope depicted in Fig. 1(a) enabled direct imaging of flowing red blood cell (RBC, red) in the blood vessels (cyan) of the buccal mucosa (Fig. 3(a)) (Visualization 1). Additionally, circulating LysM-GFP expressing immune cell (white), in the blood vessel (magenta) were successfully imaged with resident LysM-GFP+ cells (green) in the buccal mucosa (Fig. 3(b)).

 

Fig. 2. In vivo cellular-level images of buccal mucosa obtained by side-view confocal endomicroscopy. (a) CD11c-YFP cells, (b) CX3CR1-GFP cells, (c) CSF1R-GFP cells, (d) Prox1-GFP cells, (e) Thy1-YFP cells, (f) NG2-DsRed cells. Blood vessels are labeled by intravenously injected tomato lectin. Scale bars, 50 µm

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Fig. 3. Real-time fluorescence images of circulating cells in the blood vessel of mouse buccal mucosa (a) Serial images of circulating RBC (red, yellow arrowhead) in the blood vessel (cyan) in the buccal mucosa of wildtype C57BL/6N mouse. (b) Serial images of circulating LysM+ cell (white, yellow arrowhead) in the blood vessel (magenta) and resident LysM+ cells (green) in the buccal mucosa of LysM-GFP mouse. Scale bars, 50 µm.

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3.2 In vivo wide-area imaging of buccal mucosa by side-view confocal endomicroscopy

For wide-area imaging, the side-view endomicroscope could be either rotated or slid while continuously imaging the oral mucosa in the mouth of the anesthetized mouse. A circumferential mosaic image of the buccal mucosa from the roof side to the floor side in the mouth of the Prox1-GFP and NG2-DsRed double transgenic mouse was successfully obtained by rotating the side-view endomicroscope (Fig. 4(a)). Total 26 single FOV images with roughly 50% overlap were obtained and stitched. During the imaging, the position of the anesthetized mouse was adjusted by the XYZ translation stage to gently maintain contact of the side-view endomicroscope with the surface of the buccal mucosa. As shown in the magnified images, blood vessels (cyan) labeled by intravenously injected tomato lectin, Prox-1 + lymphatic vessels (yellow), and NG2 + pericytes (magenta) were simultaneously visualized at the cellular level (Fig. 4(b)).

 

Fig. 4. In vivo wide-area circumferential mosaic image of mouse buccal mucosa. (a) Mosaic images of buccal mucosa of Prox1-GFP and NG2-DsRed double transgenic mouse showing blood vessels (cyan) labeled by intravenously injected tomato lectin, Prox1 + lymphatic vessel (yellow) and NG2 + pericytes (magenta). Scale bar, 500 µm. (b) Magnified images at dotted boxes in (a). Scale bar, 50 µm.

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Next, with sliding movement of the side-view endomicroscope, a long axial mosaic image of the buccal mucosa along the surface from the mesial side to the distal side in the mouth of the CD11c-YFP and NG2-DsRed double transgenic mouse was successfully obtained (Fig. 5(a)). Total 28 single FOV images with roughly 50% overlap were obtained and stitched. Similarly, in the magnified images, individual CD11c+ cells (yellow), presumably mononuclear phagocytes including monocytes, macrophages, and dendritic cells, and NG2 + pericytes (magenta) were simultaneously visualized at the cellular level (Fig. 5(b)). Additionally, by sequentially repeating the rotation and sliding movement of the side-view endomicroscope in a zigzag path, a rectangular mosaic image of the buccal mucosa in the mouth of the NG2-DsRed mouse was obtained (Fig. 6(a)). Total 26 single FOV images with roughly 50% overlap were obtained with the zigzag movement on the buccal mucosa and then stitched and cropped. Capillary blood vessels (cyan) labeled by intravenously injected tomato lectin and NG2 + pericytes (magenta) were simultaneously visualized at the cellular level (Fig. 6(b)).

 

Fig. 5. In vivo wide-area axial mosaic image of mouse buccal mucosa. (a) Mosaic images of buccal mucosa of CD11c-YFP and NG2-DsRed double transgenic mouse showing blood vessels (cyan) labeled by intravenously injected tomato lectin, NG2 + pericytes (magenta) and CD11c+ cells (yellow). Scale bar, 500 µm. (b) Magnified images at dotted boxes in (a). Scale bar, 50 µm.

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Fig. 6. In vivo wide-area rectangular mosaic image of mouse buccal mucosa. (a) Mosaic images of buccal mucosa of NG2-DsRed mouse showing blood vessels (cyan) labeled by intravenously injected tomato lectin and NG2 + cells (yellow). (b) Magnified image at dotted box in (a). Scale bars, 50 µm.

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3.3 Repetitive imaging of the same area in buccal mucosa of a single mouse by side-view confocal endomicroscopy

Repetitive imaging of the same microscopic region of the buccal mucosa for up to 10 days was achieved by using a transgenic mouse ubiquitously expressing photoconvertible protein, Kaede [41] (Fig. 7). At day 0, naive Kaede with green fluorescence was reversibly photoconverted to red fluorescence by 405 nm laser beam illumination (180 µw for 1 minute) delivered via the side-view endomicroscope. Before the photoconversion, ubiquitously expressed Kaede with naive green fluorescence with no red fluorescence was observed. After the photoconversion, naive Kaede with green fluorescence was completely converted to red fluorescence with no residual green fluorescence. Blood vessels were clearly identified by the photoconverted red-fluorescent Kaede in endothelial cells. The photoconverted area was confined to the small area illuminated by the 405 nm laser beam, which could be easily identified by distinct red fluorescence of photoconverted Kaede out of the neighboring area with naive green fluorescence of Kaede. The photoconverted area in the buccal mucosa was longitudinally imaged by side-view confocal endomicroscopy at days 4, 7, and 10 after the photoconversion (Fig. 7). The distinct vessel branching site was marked by white arrowhead in the repetitive longitudinal imaging. At day 4, regenerated naive Kaede with green fluorescence in the muscle was observed with significantly reduced photoconverted red-fluorescent Kaede in the muscle. At day 7, the naive green-fluorescent Kaede expression in the muscle was recovered to a similar level to that observed at day 0 and the photoconverted red-fluorescent Kaede almost fully disappeared. In contrast, the recovery of naive green-fluorescent Kaede expression in the endothelial cells occurred at a later time point at day 7∼10 and the photoconverted red-fluorescent Kaede remained up to day 10, presumably due to the lower protein metabolism of endothelial cells than the muscle (Fig. 7).

 

Fig. 7. In vivo repetitive imaging of the same area in buccal mucosa. Repetitively obtained images of a same area in buccal mucosa of Kaede mouse before and after photoconversion up to 10 days. Before photoconversion, only naïve Kaede with green fluorescence was observed. After photoconversion, photoconverted Kaede with red fluorescence and regenerated Kaede with green fluorescence were observed. Scale bar, 50 µm.

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3.4 Longitudinal imaging of the buccal mucosa of a DNFB-induced oral contact allergy mouse model

Oral contact allergy was induced by repeated sensitization and challenge by 1-Fluoro-2,4-dinitrobenzene (DNFB) following the previously described protocol (Fig. 8(a)) [8]. Using the side-view confocal endomicroscope with CSF1R-GFP and LysM-GFP transgenic mice, highly dynamic changes of CSF1R+ cells and LysM+ cells in the buccal mucosa were longitudinally visualized from the sensitization stage at five days before the challenge to seven days after the challenge (Fig. 8(b)). Compared to the intact state at day -5 before the DNFB sensitization, no significant changes in the morphology and distribution of CSF1R+ cells or LysM+ cells in the buccal mucosa were observed after being sensitized twice with DNFB at day −3 (Fig. 8(b)). In contrast, at 12 hours post-challenge, a dramatic increase of CSF1R+ cells or LysM+ cells were observed (Figs. 8(b)-(d)). Interestingly, at day 1 post-challenge, the formation of a cell cluster (Fig. 5(b), asterisks) with both CSF1R+ cells and LysM+ cells were observed with no further increase in cell area (Figs. 8(c)-(d)). At day 3 post-challenge, the cell clusters disappeared and the cell areas were greatly decreased in comparison to the peak-value at 12 hours post-challenge. The distribution and total cell area of CSF1R+ cells were almost fully recovered to the baseline at the sensitization stage (Figs. 8(b)-(c)). A slight increase in the LysM+ cell area was observed but with no statistical significance (Fig. 8(d)). Finally, at day 7 post-challenge, the distribution and area of the CSF1R+ cells and LysM+ cells were almost completely recovered to the normal intact state before the DNFB challenge.

 

Fig. 8. In vivo longitudinal imaging of buccal mucosa of DNFB-induced oral contact allergy mouse model. (a) Schematic illustration of oral contact allergy induction and in vivo longitudinal endomicroscopic imaging. (b) Representative longitudinal images of the buccal mucosa in CSF1R-GFP and LysM-GFP mouse from the sensitization stage at 5 day before the challenge to 7 days after the challenge. Cell clusters formed at day 1 were marked by asterisk. Scale bar, 50 µm. (c-d) Area of CSF1R+ cells (c) or LysM+ cells (d) quantified from the longitudinal images of the buccal mucosa (number of mice ≥ 3). Statistical significances in comparison to intact state at day −5 were set at p-value less than 0.05.

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4. Discussion

We achieved longitudinal cellular-level in vivo visualization of the oral mucosa in a live mouse using a custom-designed rotatory side-view confocal endomicroscope. The side-view endomicroscope could be smoothly and non-invasively inserted into the oral cavity of the mouse, which enabled a high-resolution confocal imaging for the visualization of individual cells in the oral mucosa in vivo. Using various transgenic fluorescence reporter mice, diverse types of cells including mononuclear phagocytes, granulocytes, lymphatic endothelial cells, pericytes, and peripheral nerve cells in the buccal mucosa were successfully visualized (Fig. 2). Using the video-rate imaging capability of the imaging system used in this study [29,42], a real-time imaging of circulating cells in the blood vessel of the oral mucosa was achieved (Fig. 3). Direct imaging of a rapidly flowing RBC can be used to measure the blood flow in individual vessels and assess blood perfusion of vasculature [43], which can be useful for investigating dynamic microvascular changes in oral mucosal diseases. Additionally, a slowly rolling LysM+ cell along the vessel wall, presumably neutrophil, was visualized for over five seconds (Fig. 3(b)). In an inflamed condition, the rolling of leukocytes is greatly increased by the upregulation of adhesion molecules such as E-selectin and ICAM-1 in the vascular endothelial cells [44], which can lead to firm adhesion to the vessel wall and eventually extravasation [45]. With rotational and sliding movements of the side-view endomicroscope on the buccal mucosa, a multi-color wide-area cellular-level visualization was achieved, which can greatly increase the usability of the side-view endomicroscope to observe the large area of oral cavity (Figs. 4–6). Notably. the side-view endomicroscopy can be a useful tool to analyze these dynamic in vivo cellular behaviors triggering inflammation. We successfully performed longitudinal intravital visualization of the buccal mucosa in a DNFB-induced oral contact allergy mouse model (Fig. 8). Highly dynamic spatiotemporal cellular-level changes with pathological progression after the allergic challenge were directly monitored in a live mouse model for more than a week. It clearly revealed an acute cellular-level immune response after the induction of a contact allergy, including massive infiltration of CSF1R+ cells and LysM+ cells at 12 hours post-challenge, which was followed by the formation of a large cellular cluster at day 1 post-challenge. At three days, the clusters disappeared and the cellular level features of CSF1R+ cells and LysM+ cells in the buccal mucosa were almost completely returned to the normal intact state. These in vivo longitudinal observation results are consistent with previous studies that showed a rapid recovery after an extremely severe inflammatory reaction with abundant infiltration of immune cells into the DNFB-painted buccal mucosa in the oral mucosa after induction of a contact allergy [7,8]. Additionally, in clinical situation, oral mucosa lesions such as benign aphthous ulcers [46], keratosis and pre-malignant dysplasia, lichen planus [47] to early-stage cancer are difficult to distinguish by visual observation, so the deterministic pathology results can only be diagnosed with an invasive surgical method [48,49]. With further development for clinical adaptation, the confocal endomicroscopy capable of observing the distribution of feeding blood vessels in oral mucosa as presented in this work could be useful in predicting malignant tumors, and potentially provides additional helpful information in diagnosing various oral mucosal diseases noninvasively [50–52]. Collectively, the results presented in this work demonstrate that the proposed rotatory side-view confocal endomicroscopy could provide a useful technique to investigate the complex cellular-level pathophysiology of oral mucosal diseases.