Real-time histological evaluation of gastrointestinal tissue using non-linear microscopy

Over the past several decades, optical sectioning technologies have emerged as valuable tools for evaluating tissue histology. Unlike conventional tissue sectioning, these technologies allow for real-time intraoperative assessments and more efficient tissue triage. In the era of digital pathology, the demand for high-quality, high-throughput optical sectioning platforms is increasing, as they eliminate the need for traditional slide preparation and scanning, potentially transforming anatomical pathology workflows. While non-linear microscopy (NLM) has demonstrated promise in histological evaluation across various tissue types, its application in gastrointestinal tissue assessment remains unexplored.

This study extends the use of NLM to gastrointestinal histology and develops an image atlas to highlight its potential as an automated digital pathology platform.

Our results indicate that NLM generates diagnostic-quality images comparable to traditional H&E slides. Moreover, NLM provides valuable three-dimensional (3D) spatial information, improving clinical evaluations of key histological features such as depth of invasion, lymphovascular and perineural invasion, tumour budding and margin assessment. Time-lapse videos further demonstrate NLM's capability to capture 3D histological structures up to a depth of approximately 100 µm.

Our findings demonstrate that NLM can serve as an optical sectioning platform for gastrointestinal 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.