Transparency-enhancing technology allows the three-dimensional assessment of esophageal carcinoma obtained by endoscopic submucosal dissection

Background Although much progress has been made in diagnosis of carcinomas, no established methods have been confirmed to elucidate their morphological features. Methods Three-dimensional structure of esophageal carcinomas was assessed using transparency-enhancing technology. Endoscopically resected esophageal squamous cell carcinoma was fluorescently stained, optically cleared using a transparency-enhancing reagent called LUCID, and visualized using laser scanning microscopy. The resulting microscope images were converted to virtual HE images for observation using ImageJ software. Results Microscopic observation and image editing enabled three-dimensional image reconstruction and conversion to virtual HE images. The structure of abnormal blood vessels in esophageal carcinoma recognized by endoscopy could be observed in the 3 dimensions. Squamous cell carcinoma and normal squamous epithelium could be distinguished in the virtual HE images. Conclusions The results suggested that transparency-enhancing technology and virtual HE images may be feasible for clinical application and represent a novel histopathological method for evaluating endoscopically resected specimens. Supplementary Information The online version contains supplementary material available at 10.1007/s10388-024-01055-x.


Introduction
The Japan Esophageal Society proposed a magnifying endoscopic diagnosis of superficial esophageal carcinoma based on microvessel morphology [1].Although 3D endoscopic ultrasound enables 3D visualization of tissues, the image resolution is not sufficiently high for histopathological assessment.The ilLUmination of Cleared organs to IDentify target molecules (LUCID) method is a transparency-enhancing method using 2,2-thiodiethanol-based reagent [2][3][4].Here, we aimed to observe 3D structure of endoscopically resected esophageal carcinomas using LUCID.
The samples were imaged using a confocal microscope (FV10i-LIV; Olympus, Tokyo, Japan) and multiphotonexcited fluorescence microscope (A1MP + ; Nikon).Images of the horizontal sections from each sample were stacked and saved as single-tag image files.In this study, it took 10 s to capture a 2D image of a 500 µm square area with 10 × magnifying lens of a confocal microscope.Therefore, for example, it took about 30 min to obtain a 3D image of a 1000 µm square taken every 10 µm up to 500 µm.The image files analyzed using ImageJ (http:// rsb.info.nih.gov/ ij/) and NIS-Elements Advanced Research software (Nikon).Images of the nuclei, extracellular matrix and cytoplasm were respectively observed at 430-460 nm, 520-560 nm, and 610-630 nm.Virtual HE-stained images were created using ImageJ based on the intensity values of the scanned HE stained images.

Results
The transparency of the tissue was sufficiently enhanced for the blood vessels in the specimen to be detectable upon gross examination.Figure 1 shows a macroscopic image of an optically cleared esophagus, a fluorescent image, and virtual HE images using the procedure mentioned above.Squamous cell carcinoma (Fig. 1d, e) and normal squamous epithelium (Fig. 1f) can be distinguished in the virtual HE images.Figures 2 shows macroscopic and microscopic views of another specimen.This specimen had Type B1 and B2 vessels that were endoscopically detected.B1 vessels are defined as abnormal microvessels with a loop-like formation, whereas B2 vessels are without a loop-like formation that have elongated transformation [1].A papillary structure with B1 vessels is shown in Fig. 2c, whereas in Fig. 2i, B2 vessels showing a stretched transformation without a looplike formation can be seen in contrast to the normal intrapapillary capillary loops on the right side.
Figure 3 shows the macroscopic and microscopic views of the other specimen.This case was determined to have Type B2 vessels on endoscopy prior to ESD, and the superficial vessels on the HE-stained image alone appeared to be blood pools rather than blood vessels.However, upon transparency, the vascular structure was visible on gross examination, and the 3D reconstructed images revealed that these vessels were not strictly B2 vessels elongating from inside the tumor but rather superficial vessels pushed up into a dome shape by the tumor, mimicking B2 vessels.

Discussion
In this study, we aimed to assess the 3D structure of endoscopically resected esophageal carcinoma using a transparency-enhancing reagent that enables an arbitrary cross-section of the specimen to be observed and reconstructed in 3 dimensions, especially the 3D structure of B1 and B2 vessels.Interestingly, we found a case in which irregular vessels diagnosed as B2 vessels on endoscopy were revealed to be superficial vessels mimicking B2 vessels.The limitations of this study are the lack of quantitative evaluation, the inability to evaluate vascular invasion with virtual HE staining, and the small number of cases evaluated.The transparencyenhancing method using pathology specimens is, to the best of our knowledge, still unprecedented, so we do not believe it is immediately applicable to clinical use and is a subject for future research.However, this method could enable to more accurately assess tumor depth, vascular invasion, and margin status and detect abnormal vascular structures by

Fig. 1 Fig. 2
Fig. 1 Macroscopic and microscopic views of the optically cleared esophagus and its virtual HE image obtained from Case #1. a Macroscopic view of the optically cleared esophagus specimen obtained by ESD.This figure is a cut surface of the specimen; b fluorescent image of (a) obtained with a two-photon microscope; c-f virtual HE images converted from (b). d-f Magnified virtual HE images.d and e Are

Fig. 3
Fig. 3 Macroscopic view of the original ESD and optically cleared specimen, endoscopic view and 3D constructed images of its blood vessels, and original HE stained section obtained from Case #3. a Macroscopic view of the esophageal specimen obtained by ESD, with an white arrow indicating the direction where the tissue section was sliced using a microtome; b magnified endoscopic NBI of the yellow dotted area in (a) showing Type B2-like vessels (indicated with an white arrow); c original HE-stained image of the yellow dotted area in (a); d macroscopic view of the yellow dotted area in (a) after optical clearance, as observed from the same direction as in (c), (e) and