Probe-based three-dimensional confocal laser endomicroscopy of brain tumors: technical note

BACKGROUND: Confocal laser endomicroscopy (CLE) is used during fluorescence-guided brain tumor surgery for intraoperative microscopy of tumor tissue with cellular resolution. CLE could augment and expedite intraoperative decision-making and potentially aid in diagnosis and removal of tumor tissue. OBJECTIVE: To describe an extension of CLE imaging modality that produces Z-stack images and three-dimensional (3D) pseudocolored volumetric images. MATERIALS AND METHODS: Hand-held probe-based CLE was used to collect images from GL261-luc2 gliomas in C57BL/6 mice and from human brain tumor biopsies. The mice were injected with fluorescein sodium (FNa) before imaging. Patients received FNa intraoperatively, and biopsies were imaged immediately in the operating room. Some specimens were counterstained with acridine orange, acriflavine, or Hoechst and imaged on a benchtop confocal microscope. CLE images at various depths were acquired automatically, compiled, rendered into 3D volumes using Fiji software and reviewed by a neuropathologist and neurosurgeons. RESULTS: CLE imaging, Z-stack acquisition, and 3D image rendering were performed using 19 mouse gliomas and 31 human tumors, including meningiomas, gliomas, and pituitary adenomas. Volumetric images and Z-stacks provided additional information about fluorescence signal distribution, cytoarchitecture, and the course of abnormal vasculature. CONCLUSION: 3D and Z-stack CLE imaging is a unique new option for live intraoperative endomicroscopy of brain tumors. The 3D images afford an increased spatial understanding of tumor cellular architecture and visualization of related structures compared with two-dimensional images. Future application of specific fluorescent probes could benefit from this rapid in vivo imaging technology for interrogation of brain tumor tissue.