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

Abstract

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.

Epithelium segmentation and automated Gleason grading of prostate cancer via deep learning in label‐free multiphoton microscopic images

“…MPM has advantages of increasing the imaging depth, reducing phototoxicity and photobleaching , and the ability to generate pathology‐like images based on the natural intrinsic fluorescence properties of tissues. It has been widely used in cancer research and diagnosis, such as brain cancer , breast cancer , and esophageal cancer . In 2011, Tewari et al employed MPM to identify relevant prostatic and periprostatic structures in prostate cancer surgery.…”

Compact portable multiphoton microscopy reveals histopathological hallmarks of unprocessed lung tumor tissue in real time

“…THG/SHG/2PEF imaging is thus a promising tool for clinical intraoperative assessment of lung tumor tissue, which can lead to shorter transthoracic procedures, assessing surgical margins, acceleration of preoperative diagnostics and faster and safer CT-guided and bronchoscopic diagnostics. To further investigate the added value of this imaging technique, the quality of diagnoses by pathologists based on these images needs to be assessed, similar to the study we recently conducted for brain tumors [45], and the clinical impact of the portable THG/SHG/2PEF microscope needs to be assessed in a clinical lung cancer setting. For the diagnosing and staging of lung cancer, its impact on bronchoscopic/transthoracic procedures needs to be evaluated.…”

“…Malignant and normal human brain tissue were successfully discriminated, based on histopathological hallmarks such as increased cellularity and nuclear pleomorphism. Furthermore, our group has quantitatively detected glioma infiltration by applying automated image analysis [45,[47][48][49].…”

“…Furthermore, they found that these vesicles are associated with breast cancer growth and metastases [41–43]. In addition, our own research group used the combination of THG and SHG microscopy to detect healthy and diseased states in fresh excised brain and breast tissue [4, 44–46]. Malignant and normal human brain tissue were successfully discriminated, based on histopathological hallmarks such as increased cellularity and nuclear pleomorphism.…”

Automated interpretation of time-lapse quantitative phase image by machine learning to study cellular dynamics during epithelial–mesenchymal transition

“…As such, DHM provides additional information, which has great potential for automated interpretation of cell behavior. Also other label-free microscopic techniques have been applied to cell imaging, including harmonic generation microscopy 5 – 7 or Raman imaging. 8 These methods, however, require intense laser light passing through the specimen that could influence the cell behavior and moreover entail scanning, hence are not widefield.…”