Abstract
Background: Previous studies showed that confocal laser endomicroscopy (CLE) images of brain tumors acquired by a first-generation (Gen1) CLE system using fluorescein sodium (FNa) contrast yielded a diagnostic accuracy similar to frozen surgical sections and histologic analysis. We investigated performance improvements of a second-generation (Gen2) CLE system designed specifically for neurosurgical use.Methods: Rodent glioma models were used for in vivo and rapid ex vivo CLE imaging. FNa and 5-aminolevulinic acid were used as contrast agents. Gen1 and Gen2 CLE images were compared to distinguish cytoarchitectural features of tumor mass and margin and surrounding and normal brain regions. We assessed imaging parameters (gain, laser power, brightness, scanning speed, imaging depth, and Z-stack [3D image acquisition]) and evaluated optimal values for better neurosurgical imaging performance with Gen2.Results: Efficacy of Gen1 and Gen2 was similar in identifying normal brain tissue, vasculature, and tumor cells in masses or at margins. Gen2 had smaller field of view, but higher image resolution, and sharper, clearer images. Other advantages of the Gen2 were auto-brightness correction, user interface, image metadata handling, and image transfer. CLE imaging with FNa allowed identification of nuclear and cytoplasmic contours in tumor cells. Injection of higher dosages of FNa (20 and 40 mg/kg vs. 0.1–8 mg/kg) resulted in better image clarity and structural identification. When used with 5-aminolevulinic acid, CLE was not able to detect individual glioma cells labeled with protoporphyrin IX, but overall fluorescence intensity was higher (p < 0.01) than in the normal hemisphere. Gen2 Z-stack imaging allowed a unique 3D image volume presentation through the focal depth.Conclusion: Compared with Gen1, advantages of Gen2 CLE included a more responsive and intuitive user interface, collection of metadata with each image, automatic Z-stack imaging, sharper images, and a sterile sheath. Shortcomings of Gen2 were a slightly slower maximal imaging speed and smaller field of view. Optimal Gen2 imaging parameters to visualize brain tumor cytoarchitecture with FNa as a fluorescent contrast were defined to aid further neurosurgical clinical in vivo and rapid ex vivo use. Further validation of the Gen2 CLE for microscopic visualization and diagnosis of brain tumors is ongoing.
Highlights
Intraoperative diagnosis in neurosurgery has traditionally relied on frozen and formalin-fixed, paraffin-embedded section analysis of biopsied tissue samples
The fluorescence signal from protoporphyrin IX (PpIX) is not within the optimal detection range for these confocal laser endomicroscopy (CLE) systems, 5-aminolevulinic acid (5-ALA) is of interest in brain tumor surgery, especially for use in invasive gliomas, and our preliminary studies suggested that the PpIX signal may be faintly detected with the CLE systems
Because many imaging parameters were similar between first generation (Gen1) and second generation (Gen2) CLE, salient results are presented of observations for Gen2, and are mentioned when differences were observed with Gen1
Summary
Intraoperative diagnosis in neurosurgery has traditionally relied on frozen and formalin-fixed, paraffin-embedded section analysis of biopsied tissue samples. This technique is considered to be the “gold standard” for establishing a histopathologic diagnosis, it entails a number of significant limitations. Previous studies using a CLE system originally designed for nonneurosurgical use (e.g., gastrointestinal luminal examination) showed that a blinded review of CLE imaging of brain gliomas and meningiomas by a neuropathologist yielded an accuracy rate of 92.9%, similar to those previously reported with frozen-section analysis [4, 7,8,9]. Previous studies showed that confocal laser endomicroscopy (CLE) images of brain tumors acquired by a first-generation (Gen1) CLE system using fluorescein sodium (FNa) contrast yielded a diagnostic accuracy similar to frozen surgical sections and histologic analysis. We investigated performance improvements of a second-generation (Gen2) CLE system designed for neurosurgical use
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