Intraoperative Confocal Microscopy Research Articles

Video-Mosaicked Handheld Dual-Axis Confocal Microscopy of Gliomas: An ex vivo Feasibility Study in Humans

BackgroundIntraoperative confocal microscopy can enable high-resolution cross-sectional imaging of intact tissues as a non-invasive real-time alternative to gold-standard histology. However, all current means of intraoperative confocal microscopy are hindered by a limited field of view (FOV), presenting a challenge for evaluating gliomas, which are highly heterogeneous.ObjectiveThis study explored the use of image mosaicking with handheld dual-axis confocal (DAC) microscopy of fresh human glioma specimens.MethodsIn this preliminary technical feasibility study, fresh human glioma specimens from 6 patients were labeled with a fast-acting topical stain (acridine orange) and imaged using a newly developed DAC microscope prototype.ResultsIn comparison to individual image frames with small fields of view, mosaicked images from a DAC microscope correlate better with gold-standard H&E-stained histology images, including the ability to visualize gradual transitions from areas of dense cellularity to sparse cellularity within the tumor.ConclusionLS-DAC microscopy provides high-resolution, high-contrast images of glioma tissues that agree with corresponding H&E histology. Compared with individual image frames, mosaicked images provide more accurate representations of the overall cytoarchitecture of heterogeneous glioma tissues. Further investigation is needed to evaluate the ability of high-resolution mosaicked microscopy to improve the extent of glioma resection and patient outcomes.

SURG-04. INTRAOPERATIVE HAND-HELD LINE-SCANNED DUAL-AXIS CONFOCAL MICROSCOPY FOR VISUALIZING LOW-GRADE GLIOMAS

Abstract BACKGROUND Extent of resection is a prognostic factor for low- and high-grade gliomas. Fluorescence guided surgery (FGS) using five-aminolevulinic acid (5-ALA) is associated with greater extent of resection in high-grade gliomas. However, for low-grade gliomas, intraoperative imaging technologies such as FGS, have not been optimized to distinguish tumor from normal tissue. Intraoperative confocal microscopy can better visualize tissue cytoarchitecture in real-time. Here we report on the feasibility of a newly-developed, hand-held, line-scanned dual-axis (LS-DAC) confocal microscope in distinguishing tumor vs. normal cells in ex vivo human low-grade glioma tissue. METHOD Ten low-grade glioma patients who underwent craniotomy were enrolled. Resected specimens were labelled with acridine orange (1mM) for one minute, and exposed surfaces were immediately imaged by our device. Subsequently, specimens were cut into sections and stained with H&E for histopathological analysis. RESULTS LS-DAC confocal microscope visualized nuclei of tumor cells vs. surrounding tissue, demonstrating clear differences in cellularity in the two compartments. Acquired images were comparable to those observed on matched H&E-stained sections. Live images provided minimal motion artifact due to the higher frame rate of 16 Hz. CONCLUSION LS-DAC confocal microscope provided real-time, high-contrast mosaic images of human low-grade glioma tissue ex vivo. The ability to distinguish tumor vs. normal tissue at the cellular level with little motion artifact, as well as the device’s hand-held design, suggests this technology merits additional investigation as an intraoperative adjunct for low-grade glioma surgery.

In vivo intraoperative confocal microscopy for real-time histopathological imaging of brain tumors

Frozen-section analysis is the current standard for the intraoperative diagnosis of brain tumors. Intraoperative confocal microscopy is an emerging technology with the potential to visualize tumor histopathological features and cell morphology in real time. The authors report their findings using this new intraoperative technology in vivo with sodium fluorescein contrast during the course of 50 microsurgical tumor resections. Eighty-eight regions were visualized with confocal microscopy, and corresponding biopsy samples were examined with routine neuropathological analysis. The tumors studied included meningiomas, schwannomas, gliomas of various grades, and a hemangioblastoma. The confocal microscopic features of each tumor and of various artifacts inherent to the technology were documented. A pathologist working in a blinded fashion reviewed a subset of the images in a further evaluation of the usefulness of the device as a diagnostic tool. Overall, intraoperative confocal imaging correlated surprisingly well with corresponding traditional histological findings, including the identification of many pathognomonic cytoarchitectural features of various brain tumors. In the blinded study, 26 (92.9%) of 28 lesions were diagnosed correctly. Further study will be necessary for better definition of the role of intraoperative confocal microscopy as a routine adjunct for intraoperative brain tumor diagnosis.

Confocal Neurolasermicroscopy

To the Editor: We read with interest the article by Sanai et al1 that reports the use of intraoperative confocal microscopy in human brain tumors for their prospective feasibility study. The authors stated that this exciting technology has not been applied in human brain tissue or pathology so far. However, 2 years ago, the exact same technology was demonstrated for the first time in an approach in human glioblastoma multiforme to generate high-resolution images after topical staining of the resected specimens. In this previous pilot study from 2009,2 a major observation was that brain tumor areas were clearly detectable with this new technique, which we at that time called neurolasermicroscopy. Although exactly the same confocal scanning device was used in both studies mentioned, the way of staining the tissue of interest was slightly different. In the ex vivo approach, topically applied acriflavine hydrochloride was administered onto the surface of the tissue and allowed to reach the diagnosis by determination of World Health Organization criteria for glioblastoma multiforme. However, in the present study, intravenous fluorescein was injected during ongoing neurosurgery and distinguished primarily the blood vessels without characterizing important cellular details such as mitotic figures or pseudopalisading necrosis. In the fascinating field of high-resolution imaging at the cellular and subcellular levels, there are many problems ahead that should be solved in the future when techniques like neurolasermicroscopy are adapted for the operating room.3 The administration of effective and safe contrast agents is one essential step, as mentioned above. The reprocessing of the highly sensitive confocal laser scanning device is another crucial step toward clinical application.

Intraoperative confocal microscopy in the visualization of 5-aminolevulinic acid fluorescence in low-grade gliomas

Greater extent of resection (EOR) for patients with low-grade glioma (LGG) corresponds with improved clinical outcome, yet remains a central challenge to the neurosurgical oncologist. Although 5-aminolevulinic acid (5-ALA)-induced tumor fluorescence is a strategy that can improve EOR in gliomas, only glioblastomas routinely fluoresce following 5-ALA administration. Intraoperative confocal microscopy adapts conventional confocal technology to a handheld probe that provides real-time fluorescent imaging at up to 1000× magnification. The authors report a combined approach in which intraoperative confocal microscopy is used to visualize 5-ALA tumor fluorescence in LGGs during the course of microsurgical resection. Following 5-ALA administration, patients with newly diagnosed LGG underwent microsurgical resection. Intraoperative confocal microscopy was conducted at the following points: 1) initial encounter with the tumor; 2) the midpoint of tumor resection; and 3) the presumed brain-tumor interface. Histopathological analysis of these sites correlated tumor infiltration with intraoperative cellular tumor fluorescence. Ten consecutive patients with WHO Grades I and II gliomas underwent microsurgical resection with 5-ALA and intraoperative confocal microscopy. Macroscopic tumor fluorescence was not evident in any patient. However, in each case, intraoperative confocal microscopy identified tumor fluorescence at a cellular level, a finding that corresponded to tumor infiltration on matched histological analyses. Intraoperative confocal microscopy can visualize cellular 5-ALA-induced tumor fluorescence within LGGs and at the brain-tumor interface. To assess the clinical value of 5-ALA for high-grade gliomas in conjunction with neuronavigation, and for LGGs in combination with intraoperative confocal microscopy and neuronavigation, a Phase IIIa randomized placebo-controlled trial (BALANCE) is underway at the authors' institution.

Intraoperative Confocal Microscopy for Brain Tumors: A Feasibility Analysis in Humans

The ability to diagnose brain tumors intraoperatively and identify tumor margins during resection could maximize resection and minimize morbidity. Advances in optical imaging enabled production of a handheld intraoperative confocal microscope. To present a feasibility analysis of the intraoperative confocal microscope for brain tumor resection. Thirty-three patients with brain tumor treated at Barrow Neurological Institute were examined. All patients received an intravenous bolus of sodium fluorescein before confocal imaging with the Optiscan FIVE 1 system probe. Optical biopsies were obtained within each tumor and along the tumor-brain interfaces. Corresponding pathologic specimens were then excised and processed. These data was compared by a neuropathologist to identify the concordance for tumor histology, grade, and margins. Thirty-one of 33 lesions were tumors (93.9%) and 2 cases were identified as radiation necrosis (6.1%). Of the former, 25 (80.6%) were intra-axial and 6 (19.4%) were extra-axial. Intra-axial tumors were most commonly gliomas and metastases, while all extra-axial tumors were meningiomas. Among high-grade gliomas, vascular neoproliferation, as well as tumor margins, were identifiable using confocal imaging. Meningothelial and fibrous meningiomas were distinct on confocal microscopy--the latter featured spindle-shaped cells distinguishable from adjacent parenchyma. Other tumor histologies correlated well with standard neuropathology tissue preparations. Intraoperative confocal microscopy is a practicable technology for the resection of human brain tumors. Preliminary analysis demonstrates reliability for a variety of lesions in identifying tumor cells and the tumor-brain interface. Further refinement of this technology depends upon the approval of tumor-specific fluorescent contrast agents for human use.

Real Time Intraoperative Confocal Laser Microscopy-Guided Surgery

To assess the potential utility of in vivo histologic surface and subsurface imaging in real-time using the Optiscan confocal laser microscope to detect diseased tissue at the time of surgery. The goal of surgical treatment of diseases such as cancer is complete microscopic resection of diseased tissue; however, current methods for the assessment of extent of disease at the time of surgery are inadequate. We assessed the potential of the Optiscan confocal laser microscope, a new device developed for real-time in vivo histologic surface and subsurface imaging during surgery. Intravenous Fluorescein Sodium contrast enabled visualization of cellular and architectural morphology of intra-abdominal organs with magnification equivalent to light microscopy and enabled differentiation between normal organs and disease. Real time intraoperative confocal microscopy has significant potential application in detecting disease, and influencing decision-making at the time of surgery.