Label-free imaging of human brain tissue at subcellular resolution for potential rapid intra-operative assessment of glioma surgery.

Background: Frozen section and smear preparation are the current standard for intraoperative histopathology during cancer surgery. However, these methods are time-consuming and subject to limited sampling. Multiphoton microscopy (MPM) is a high-resolution non-destructive imaging technique capable of optical sectioning in real time with subcellular resolution. In this report, we systematically investigated the feasibility and translation potential of MPM for rapid histopathological assessment of label- and processing-free surgical specimens. Methods: We employed a customized MPM platform to capture architectural and cytological features of biological tissues based on two-photon excited NADH and FAD autofluorescence and second harmonic generation from collagen. Infiltrating glioma, an aggressive disease that requires subcellular resolution for definitive characterization during surgery, was chosen as an example for this validation study. MPM images were collected from resected brain specimens of 19 patients and correlated with histopathology. Deep learning was introduced to assist with image feature recognition. Results: MPM robustly captures diagnostic features of glioma including increased cellularity, cellular and nuclear pleomorphism, microvascular proliferation, necrosis, and collagen deposition. Preliminary application of deep learning to MPM images achieves high accuracy in distinguishing gray from white matter and cancer from non-cancer. We also demonstrate the ability to obtain such images from intact brain tissue with a multiphoton endomicroscope for intraoperative application. Conclusion: Multiphoton imaging correlates well with histopathology and is a promising tool for characterization of cancer and delineation of infiltration within seconds during brain surgery.

Optical Characterization of Neurosurgical Operating Microscopes: Quantitative Fluorescence and Assessment of PpIX Photobleaching.

Protoporphyrin IX (PpIX) induced by 5-aminolevulinic acid (5-ALA) is increasingly used as a fluorescent marker for fluorescence-guided resection of malignant gliomas. Understanding how the properties of the excitation light source and PpIX fluorescence interact with the surgical microscope is critical for effective use of the fluorescence-guided tumor resection technique. In this study, we performed a detailed assessment of the intensity of the emitted blue light and white light and the light beam profile of clinical grade operating microscopes used for PpIX visualization. These measurements revealed both recognized fluorescence photobleaching limitations and unrecognized limitations that may alter quantitative observations of PpIX fluorescence obtained with the operating microscope with potential impact on research and clinical uses. We also evaluated the optical properties of a photostable fluorescent standard with an excitation-emission profile similar to PpIX. In addition, we measured the time-dependent dynamics of 5-ALA-induced PpIX fluorescence in an animal glioma model. Finally, we developed a ratiometric method for quantification of the PpIX fluorescence that uses the photostable fluorescent standard to normalize PpIX fluorescence intensity. This method increases accuracy and allows reproducible and direct comparability of the measurements from multiple samples.

TM1-IR680 peptide for assessment of surgical margin and lymph node metastasis in murine orthotopic model of oral cancer.

Treatment outcome after surgical removal in oral carcinoma is poor due to inadequate methodologies available for marking surgical margins. Even though some methodologies for intraoperative margin assessment are under clinical and preclinical trials for other solid tumours, a promising modality for oral cancer surgery is not developed. Fluorescent-based optical imaging using Near Infrared (NIR) dyes tagged to tumour specific target will be an optimal tool for this purpose. One such target, Gastrin Releasing Peptide Receptor (GRPR) was selected for the study, and its binding peptide, TM1-IR680, was tested for its efficacy for surgical margin prediction in murine orthotopic model of oral cancer, derived from primary samples. Here, for the first time in a preclinical analysis, we show that the size and margin of oral cancer can be predicted, as revealed by 3D-imaging. Interestingly, the peptide was sensitive enough to detect lymph nodes that harboured dispersed tumour cells before colonization, which was impossible to identify by conventional histopathology. We recommend the use of TM1-NIR dyes alone or in combination with other technologies to improve the clinical outcome of oral cancer surgery.

Preclinical assessment of a 980-nm diode laser ablation system in a large animal tumor model.

PURPOSE: To characterize the performance of a 980-nm diode laser ablation system in an in vivo tumor model. MATERIALS AND METHODS: This study was approved by the institutional animal care and use committee. The ablation system consisted of a 15-W, 980-nm diode laser, flexible diffusing-tipped fiber optic, and 17-gauge internally cooled catheter. Ten immunosuppressed dogs were inoculated subcutaneously with canine-transmissible venereal tumor fragments in eight dorsal locations. Laser ablations were performed at 79 sites where inoculations were successful (99%) at powers of 10 W, 12.5 W, and 15 W, with exposure times between 60 and 180 seconds. In 20 cases, multiple overlapping ablations were performed. After the dogs were euthanized, the tumors were harvested, sectioned along the applicator tract, measured, and photographed. Measurements of ablation zone were performed on gross specimen. Histopathology and viability staining was performed with hematoxylin and eosin and nicotinamide adenine dinucleotide hydrogen staining. RESULTS: Gross pathologic examination confirmed a well circumscribed ablation zone with sharp boundaries between thermally ablated tumor in the center surrounded by viable tumor tissue. When a single applicator was used, the greatest ablation diameters ranged from 12 mm at the lowest dose (10 W, 60 seconds) to 26 mm at the highest dose (15 W, 180 seconds). Multiple applicators created ablation zones as large as 42 mm in greatest diameter (with the lasers operating at 15 W for 120 seconds). CONCLUSIONS: The new 980-nm diode laser and internally cooled applicator effectively create large ellipsoid thermal ablations in less than 3 minutes.

Radiofrequency ablation: post-ablation assessment using CT perfusion with pharmacological modulation in a rat subcutaneous tumor model.

RATIONALE AND OBJECTIVES: Inflammatory reaction surrounding the ablated area is a major confounding factor in the early detection of viable tumor after radiofrequency (RF) ablation. A difference in the responsiveness of normal and tumor blood vessels to vasoactive agents may be used to distinguish these regions in post-ablation follow-up. The goal of this study was to examine longitudinal perfusion changes in untreated viable tumor and the peripheral hyperemic rim of RF-ablated tumor in response to a vasoconstrictor (phenylephrine) or vasodilator (hydralazine) in a subcutaneous rat tumor model. MATERIALS AND METHODS: Bilateral subcutaneous shoulder tumors were inoculated in 24 BDIX rats and evenly divided into two groups (phenylephrine and hydralazine groups). One tumor in each animal was completely treated with RF ablation (at 90 +/- 2 degrees C for 3 minutes), and the other remained untreated. Computed tomographic perfusion scans before and after phenylephrine (10 microg/kg) or hydralazine (5 mg/kg) administration were performed 2, 7, and 14 days after ablation. Four rats per group were euthanized on each scan day, and pathologic evaluation was performed. The changes of blood flow in the peripheral rim of ablated tumor and untreated viable tumor in response to phenylephrine or hydralazine at each time point were compared. The diagnostic accuracy of viable tumor using the percentage change of blood flow in response to phenylephrine and hydralazine was compared using receiver-operating characteristic analysis. RESULTS: The peripheral rim of ablated tumor presented with a hyperemic reaction with dilated vessels and congestion on day 2 after ablation, numerous inflammatory vessels on day 7, and granulation tissue formation on day 14. Phenylephrine significantly decreased the blood flow in the peripheral hyperemic rim of ablated tumor on days 2, 7, and 14 by 16.3 +/- 9.7% (P = .001), 24.0 +/- 22.6% (P = .007), and 31.1 +/- 25.4% (P = .045), respectively. In untreated viable tumor, the change in blood flow after phenylephrine was irregular and insignificant. Hydralazine decreased the blood flow in the peripheral rim of both ablated tumor and untreated viable tumor. Receiver-operating characteristic analysis showed that reliable tumor diagnosis using the percentage change of blood flow in response to phenylephrine was noted on days 2 and 7, for which the areas under the curve were 0.82 (95% confidence interval, 0.64-1.00) and 0.81 (95% confidence interval, 0.56-1.00), respectively. However, tumor diagnosis using the blood flow change in response to hydralazine was unreliable. CONCLUSION: Phenylephrine markedly decreased blood flow in the peripheral hyperemic rim of ablated tumor but had little effect on the untreated viable tumor. Computed tomographic perfusion with phenylephrine may be useful in the long-term treatment assessment of RF ablation.

Radio-frequency ablation of VX2 rabbit tumors: assessment of completeness of treatment by using contrast-enhanced harmonic power Doppler US.

PURPOSE: To assess contrast material-enhanced harmonic power Doppler and fundamental color Doppler ultrasonography (US) in the detection of residual viable tumor tissue after radio-frequency (RF) ablation in tumors embedded in fat. MATERIALS AND METHODS: Twenty-eight VX2 tumors were implanted into the retroperitoneum of 14 rabbits. Tumors were examined with contrast-enhanced fundamental color Doppler US and harmonic power Doppler US before and 10 minutes after RF ablation. Saline-enhanced RF ablation (30 mL/h) was performed over 10 minutes with 28-W RF power. Follow-up included repeat US examinations. Necropsies and histopathologic assessment were performed after detection of residual untreated tumor at US or 3 weeks after ablation. RESULTS: VX2 tumors reached a mean size of 21 mm +/- 9 (SD) (size range, 6-43 mm) 25 days after implantation. All tumors larger than 31 mm showed signs of central necrosis at US. Before ablation, intense vascularity was detected in all tumors with both contrast-enhanced US modes. Histopathologic assessment at the end of the follow-up period revealed local relapses due to incomplete ablation in 14 (50%) of 28 cases. Detection of residual tumor was missed in all cases with contrast-enhanced color Doppler US. Contrast-enhanced harmonic power Doppler US depicted residual flow in 12 of the 14 cases (sensitivity, 86%) in which local relapses occurred. There was a significant (P <.005, McNemar test) improvement in detection of residual tumor when the harmonic power Doppler mode was used. CONCLUSION: Contrast-enhanced harmonic power Doppler US has greater sensitivity than contrast-enhanced color Doppler US for detecting residual VX2 tumor following ablation. Therefore, contrast-enhanced harmonic power Doppler US may be a useful additional method for the detection of residual tumors after RF ablation.