Efficacy and Feasibility of Tissue-clearing Technique and Three-dimensional Imaging in the Human Gastrointestinal Tissues Using LUCID.

Abstract

Introduction: The applicability of tissue-clearing technology and 3D imaging in human specimens has not been fully investigated. We investigated the effectiveness and feasibility of the ilLUminate Cleared organs to IDentify target molecules (LUCID) protocol using human gastrointestinal specimens.

Methods: The gastrointestinal mucosa specimens resected via ESD including the esophagus, stomach, duodenum, and large intestine were fluorescently stained and optically cleared using LUCID. Cleared specimens were imaged three-dimensionally by confocal laser scanning microscope, and the observable depth at any five points was measured and compared to non-cleared specimens, respectively. After clearing and imaging, the specimens were restored to the PPFE form again, and conventional two-dimensional pathological evaluation using hematoxylin-eosin (HE), Ki67 (MIB-1), p53, and E-cadherin staining was performed to compare them with their pre-clearing state.

Results: The observable depth was significantly extended after clearing for specimens from each organ (esophagus 228.3±14.9 µm vs. 1036.7±62.9 µm, p<0.05; stomach 115.2±5.5 µm vs. 428.7±15.9 µm, p<0.05; duodenum 256.2±9.5 µm vs. 787.0±18.6 µm, p<0.05, large intestine 113.9±5.4 µm vs. 436.6±18.5 µm, p<0.05). The pathological evaluation after clearing revealed a preserved fine structure and staining and showed no apparent deformation, degeneration, or tissue damage compared with before clearing.

Conclusions: The effectiveness of tissue clearing using LUCID on human gastrointestinal specimens was demonstrated, and the LUCID protocol had minimal impact on specimen morphology and staining. Although many limitations and issues remain, LUCID is expected to be a method that enables comprehensive analysis of the three-dimensional structure of human gastrointestinal mucosa and lesions.