Dye‐Enhanced Multimodal Confocal Imaging as a Novel Approach to Intraoperative Diagnosis of Brain Tumors

Abstract

Intraoperative diagnosis plays an important role in accurate sampling of brain tumors, limiting the number of biopsies required and improving the distinction between brain and tumor. The goal of this study was to evaluate dye-enhanced multimodal confocal imaging for discriminating gliomas from nonglial brain tumors and from normal brain tissue for diagnostic use. We investigated a total of 37 samples including glioma (13), meningioma (7), metastatic tumors (9) and normal brain removed for nontumoral indications (8). Tissue was stained in 0.05 mg/mL aqueous solution of methylene blue (MB) for 2-5 minutes and multimodal confocal images were acquired using a custom-built microscope. After imaging, tissue was formalin fixed and paraffin embedded for standard neuropathologic evaluation. Thirteen pathologists provided diagnoses based on the multimodal confocal images. The investigated tumor types exhibited distinctive and complimentary characteristics in both the reflectance and fluorescence responses. Images showed distinct morphological features similar to standard histology. Pathologists were able to distinguish gliomas from normal brain tissue and nonglial brain tumors, and to render diagnoses from the images in a manner comparable to haematoxylin and eosin (H&E) slides. These results confirm the feasibility of multimodal confocal imaging for intravital intraoperative diagnosis.

Ex vivo confocal microscopy imaging to identify tumor tissue on freshly removed brain sample

“…In the field of brain tumors, confocal microscopy has been used for imaging purpose of brain tumors in vivo on murine models [5,6]. In studies involving humans in vivo and ex vivo confocal microscopy has been used [7]. The main difference with in vivo studies is the use of acridine orange instead of methylene blue staining [5,6].…”

Volumetric imaging and quantification of cytoarchitecture and myeloarchitecture with intrinsic scattering contrast

“…Third-harmonic generation also visualizes neuronal cell bodies due to lack of structural phase matching [5]. Elastic backscattering techniques such as Optical Coherence Microscopy (OCM) and confocal reflectance microscopy provide contrast comparable to third-harmonic generation in brain tissue, depicting both neuronal cell bodies and myelinated axons [6][7][8][9]. The birefringent properties of the myelin sheath can be interrogated with polarization-sensitive imaging to provide additional contrast [10].…”

Comparative evaluation of methylene blue and demeclocycline for enhancing optical contrast of gliomas in optical images

“…Pathologists and neuropathologists, in particular, were successful in differentiating primary brain tumors from metastases and meningioma. 4 To compare DMN and MB staining, in Fig. 3, we show images of the two halves of a GBM sample, stained with DMN and MB.…”

“…3,4 For confocal imaging of DMN-stained samples, the imaging system was modified to allow for the excitation of DMN and detection of DMN fluorescence signal. In particular, a 402-nm diode laser (MicroLaser Systems, Garden Grove, California, USA) was used as the light source.…”