Confocal laser endomicroscopy (CLE) allows intraoperative “optical biopsy” at a cellular level without tissue processing. We report the evolution of this technology and present analysis of recent use of the updated CLE tool on patients during fluorescein sodium (FNa) guided brain tumor surgeries. Our clinical experience with CLE includes 237 patients with gliomas, meningiomas and other CNS pathologies examined ex vivo and in vivo using a Generation1 CLE and 48 patients using a Generation2 CLE (19 HGG, 3 LGG, 11 pituitary adenomas, 2 craniopharyngiomas, 2 metastases, 2 schwannomas, 4 meningiomas, 2 treatment effects, 1 focal cortical dysplasia, 2 hemangioblastomas) examined ex vivo. Acriflavine (AF) and acridine orange (AO) were used ex vivo on selected tissue samples. In vivo CLE during FNa-guided surgery produced 77.7 ± 46.2 (average) images/optical biopsy location. A first diagnostic image was identified within seconds of CLE application. In vivo CLE specificity/sensitivity (FNa) was equal or better than frozen section (94%/91% gliomas, 93%/97% meningiomas respectively). Generation2 CLE showed improved image resolution and system operation for detectable tumor signal with Z-stack 3D imaging compared to Generation1. FNa 2 mg/kg administered during induction of anesthesia was sufficient for wide field tumor fluorescence visualization using the operative microscope Yellow560 mode. However, additional injection of FNa (2–5 mg/kg) before optical biopsy was necessary to provide sufficient CLE image contrast for immediate ex vivo CLE imaging in most of the cases. CLE imaging after rapid ex vivo application of AF and AO revealed more intense and specific contrasted intracellular structural patterns, such as nuclei. Overall, CLE rapidly provided information on tissue architecture and atypical cellular features and has potential to improve the surgery-pathology workflow. Additional injection of FNa during fluorescence-guidance surgery may be necessary for CLE optical biopsy, which may interfere with the operative microscope wide field fluorescence visualization, and require further investigation.
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