Over the past several decades, optical sectioning technologies have emerged as valuable tools for evaluating tissue histology. Because these techniques eliminate the need for laborious paraffin embedding and microtome sectioning, they may enable real-time intraoperative assessments and more efficient tissue triaging. This study explores the use of one such technique, nonlinear microscopy (NLM) in the context of gastrointestinal (GI) pathology. We provide a comprehensive NLM-based atlas of GI pathology, consisting of video clips demonstrating real-time analysis and three-dimensional (3D) scanning capabilities. NLM provides valuable 3D spatial information, improving clinical evaluations of key histological features such as depth of invasion, lymphovascular and perineural invasion, tumor budding, and margin assessment. Time-lapse videos further demonstrate NLM's capacity to capture 3D histological structures up to a depth of approximately 100 µm. Our findings indicate that NLM can serve as an optical sectioning platform for GI histology, providing both diagnostic-quality imaging and advanced 3D visualisation. The introduction of an NLM-based atlas has the potential to redefine anatomical pathology workflows and advance digital pathology image analysis.

Fresh or fixed gastrointestinal surgical specimens were collected under an IRB-approved study protocol. Specimens were dissected into approximately 2 × 1 × 0.5 cm sections and stained directly with acridine orange and sulforhodamine 101, labeling nucleic acids and proteins, respectively. Stained tissues were briefly rinsed and mounted on a custom-designed inverted microscope stage. Video-rate nonlinear microscopy was performed with excitation wavelength of 1030 nm and a 20×/0.75 NA dry objective lens. A macro image of the entire stage was acquired using a sub-stage camera (shown on the right in the video clips). A surgical pathologist performed real-time imaging by navigating with a motorized stage and an objective lens scanner.