Progress in Confocal Laser Endomicroscopy for Neurosurgery and Technical Nuances for Brain Tumor Imaging With Fluorescein

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.

Ex Vivo Fluorescein-Assisted Confocal Laser Endomicroscopy (CONVIVO® System) in Patients With Glioblastoma: Results From a Prospective Study

“…With this study we were also interested in categorizing some morphological patterns: cellular features and tumor structures, such as pleomorphism, atypia, hypercellularity, and neovascularization appeared to correlate with the matched permanent sections and known tissue architecture. Although quality of images was not as good as the one shown in the paper by Belykh and colleagues (15), CONVIVO ® ex vivo scanning permitted to clearly identify GBM tissue during surgery, leading to an intraoperative correct diagnosis in a high percentage of cases, fulfilling the primary objective in such study. As a matter of fact, our protocol permitted to the pathologist to analyze CONVIVO ® tissue samples in a blinded manner, never knowing anticipately the results from permanent or frozen section images.…”

“…This limitation could be partially overcome by a totally in vivo setting, which needs a dedicated sterile sheath covering the CONVIVO ® probe, allowing for a direct tumor bed analysis, surely much closer to the SF injection time. As a matter of fact, in previous in vivo published series, performed with other confocal prototypes, or with CONVIVO ® system only in animals (15,19), the quality of the image related to the improved background fluorescence, is much higher. In addition, other authors suggested to use different protocols of SF injection, right before in vivo CLE analysis, with an impact on image quality (6,13).…”

“…Although such technique is not available yet on a routine basis, its utilization could improve tumor visualization at the tumor margin and quicken intraoperative diagnosis, since the machinery could be used directly in the OR. In particular, few works have studied specificity and sensitivity of first-generation CLE to provide diagnostic information during biopsy or resection of human brain tumors, finding comparable values to frozen sections (6,(13)(14)(15)(16)(17). Less data are currently available on CLE analysis at human HGGs margins (8,18).…”

Confocal Laser Endomicroscopy Assessment of Pituitary Tumor Microstructure: A Feasibility Study

“…Wide-field fluorescence imaging relies on visualization of unspecific metabolic or targeted fluorescent molecular probes as discussed above; this imaging technique is advantageous for margin detection. Confocal imaging involves a point-scanning excitation laser and a pinhole aperture that filters out-of-focus detected light that allows pixel-by-pixel en face imaging of tissue at a certain depth with submicron resolution [ 35 ]. Various technologies that employ the confocal principle include confocal reflectance microscopy, which relies on the detection of reflected excitation light, and confocal fluorescence microscopy, which relies on the detection of a fluorescence signal of specific wavelengths emitted by a fluorophore.…”

Blood-Brain Barrier, Blood-Brain Tumor Barrier, and Fluorescence-Guided Neurosurgical Oncology: Delivering Optical Labels to Brain Tumors

“…FLS-guided resection of high-grade glioma was successful in a recent Phase II clinical trial (n = 46 patients) (234) and in a prospective observational study (n = 279 patients) (78), indicating its clinical applicability. FLS has also been used as a contrast for intraoperative assessment of tissue microstructure using confocal laser endomicroscopy (235,236).…”