Multiphoton Microscopy: A Feasability Study to Explore the Potential Intraoperative Tool for TURBT Diagnosis

Background: Multiphoton microscopy (MPM) is a nonlinear optical imaging technology, allowing process-free imaging of fresh tissue up to a depth of 0.5 mm, with a combined signal of autofluorescence and second harmonic generation. The absence of bladder muscle in a transurethral resection of bladder tumor (TURBT) increases risk of residual disease, recurrence, and understaging, and an intraoperative assessment of resection depth and margins may avoid unnecessary second TURBTs. Objective: The objective of this study was to test the feasibility of MPM in the real-time detection of bladder muscle in cold cup biopsies of patients undergoing TURBT. Methods: This was a prospective feasibility study. Cold cup biopsies were taken intraoperatively from the resection bed during TURBT. Tissue was stored in phosphate buffered saline solution combined with fluorescent dyes (orange acridine and rhodamine). MPM imaging of the tissue was performed first, with z stack images of 100 µm maximum depth for 3D imaging. After MPM imaging, tissue was fixed in formalin and sent to the biorepository for standard histopathology. A descriptive analysis was performed between MPM tissue detection and final histopathology results. Results: Ten patients were included in this pilot study. Tissue was properly imaged for all patients, although 3 of them had artifacts, and we detected 5 times muscularis propria on MPM imaging. MPM average procedure time from biopsy to formalin fixation (MPM imaging) was 16.2 minutes (12-20 minutes). On final pathology, resection depth was correctly performed for only 4 patients of 10 (resection including bladder muscle). We found an 80% sensitivity on resection depth between first layer identified on MPM and final pathology (8/10 patients) and 100% correlation with final histopathology in specimens for the assessment of detrusor muscle. Conclusions: This study supports the use of MPM as a novel intraoperative tool which can evaluate cold cup biopsies during TURBT for resection depth and potentially avoid unnecessary second look procedure.