Quantitative Third Harmonic Generation Microscopy for Assessment of Glioma in Human Brain Tissue

• Published in: Advanced science 2019-06, Vol.6 (11), p.1900163-n/a
• Main Authors: Zhang, Zhiqing, de Munck, Jan C., Verburg, Niels, Rozemuller, Annemieke J., Vreuls, Willem, Cakmak, Pinar, van Huizen, Laura M. G., Idema, Sander, Aronica, Eleonora, de Witt Hamer, Philip C., Wesseling, Pieter, Groot, Marie Louise
• Format: Article
• Language: English
• Subjects: Automation; Brain cancer; Epilepsy; glioma infiltration; label‐free microscopy; Lipids; Magnetic resonance imaging; Medical imaging; Medical prognosis; Microscopy; Morphology; neuropathology; neurosurgery; NMR; Nuclear magnetic resonance; Patients; Scandals; Spectrum analysis; Statistical analysis; Surgical outcomes; third harmonic generation; Tomography; Tumors
• ISSN: 2198-3844; 2198-3844
• DOI: 10.1002/advs.201900163

Distinguishing tumors from normal brain cells is important but challenging in glioma surgery due to the lack of clear interfaces between the two. The ability of label‐free third harmonic generation (THG) microscopy in combination with automated image analysis to quantitatively detect glioma infiltration in fresh, unprocessed tissue in real time is assessed. The THG images reveal increased cellularity in grades II–IV glioma samples from 23 patients, as confirmed by subsequent hematoxylin and eosin histology. An automated image quantification workflow is presented for quantitative assessment of the imaged cellularity as a reflection of the degree of glioma invasion. The cellularity is validated in three ways: 1) Quantitative comparison of THG imaging with fluorescence microscopy of nucleus‐stained samples demonstrates that THG reflects the true tissue cellularity. 2) Thresholding of THG cellularity differentiates normal brain from glioma infiltration, with 96.6% sensitivity and 95.5% specificity, in nearly perfect (93%) agreement with pathologists. 3) In one patient, a good correlation between THG cellularity and preoperative magnetic resonance and positron emission tomography imaging is demonstrated. In conclusion, quantitative real‐time THG microscopy accurately assesses glioma infiltration in ex vivo human brain samples, and therefore holds strong potential for improving the accuracy of surgical resection. The intraoperative detection of brain tumor boundaries is a long standing problem in the clinic. Current clinical imaging techniques cannot accurately localize the real tumor boundaries because of insufficiently high resolution, quality of staining, and/or lack of real‐time imaging. Label‐free third harmonic generation microscopy is shown to be able to detect tumor boundaries quantitatively, via direct imaging of cellularity, in real time.