Label-Free Neurosurgical Pathology with Stimulated Raman Imaging

Abstract

The goal of brain tumor surgery is to maximize tumor removal without injuring critical brain structures. Achieving this goal is challenging as it can be difficult to distinguish tumor from nontumor tissue. While standard histopathology provides information that could assist tumor delineation, it cannot be performed iteratively during surgery as freezing, sectioning, and staining of the tissue require too much time. Stimulated Raman scattering (SRS) microscopy is a powerful label-free chemical imaging technology that enables rapid mapping of lipids and proteins within a fresh specimen. This information can be rendered into pathology-like images. Although this approach has been used to assess the density of glioma cells in murine orthotopic xenografts models and human brain tumors, tissue heterogeneity in clinical brain tumors has not yet been fully evaluated with SRS imaging. Here we profile 41 specimens resected from 12 patients with a range of brain tumors. By evaluating large-scale stimulated Raman imaging data and correlating this data with current clinical gold standard of histopathology for 4,422 fields of view, we capture many essential diagnostic hallmarks for glioma classification. Notably, in fresh tumor samples, we observe additional features, not seen by conventional methods, including extensive lipid droplets within glioma cells, collagen deposition in gliosarcoma, and irregularity and disruption of myelinated fibers in areas infiltrated by oligodendroglioma cells. The data are freely available in a public resource to foster diagnostic training and to permit additional interrogation. Our work establishes the methodology and provides a significant collection of reference images for label-free neurosurgical pathology. Cancer Res; 76(12); 3451-62. ©2016 AACR.

Fig. 1. Label-free chemical imaging of human brain with stimulated Raman scattering (SRS) microscopy

(A) Energy diagrams of stimulated Raman scattering (SRS) and spontaneous Raman scattering. In spontaneous Raman scattering, a small amount of photons are shifted in energy from the laser frequency due to their interaction with the vibrational levels of molecules in the sample. The energy shift is defined as Raman shift, which is often reported in wavenumbers (cm⁻¹). Note that spontaneous Raman scattering is typically very weak. SRS occurs when the sample is excited by synchronized pump and Stokes beams of ultrafast lasers. The frequency difference of the two laser beams defines the Raman shift used for imaging. SRS signals are much stronger than spontaneous Raman scattering due to the stimulated emission process. (B) Schematic representation of SRS microscopy (upper), and representative two-color SRS imaging of fresh brain tissue at 2940 and 2854 cm⁻¹ (below). (C) SRS spectra of brain tumor, white matter, gray matter, and necrotic tissue show distinct spectral features. (D) SRS imaging of a fresh human brain tissue sample at the white matter and gray matter junction (green, lipid; blue, protein). White matter is featured by strong lipid signals mainly from the myelin sheath. Gray matter contains lower lipid composition, showing in dark blue. Scale bar, 100 μm. (E) SRS imaging (left) of frozen normal human brain tissue sections from an autopsy case (case A1). SRS imaging could rapidly and clearly identify white and gray matter based on the lipid/protein contrast. In contrast, H&E staining images (middle) did not provide a clear distinction between white and gray matter. White matter was confirmed by LFB staining (right). (F) Zoom-in images in (E) show very fine structures of single myelin fibers at the transition area from white matter to gray matter. (G) A large-scale image of white/gray matter (~7 mm in length) and the intensity profile (sum along the vertical direction) shows that lipid content in the white matter is higher than that of the gray matter by ~2 fold. Scale bars: (D and F), 100 μm; (E and G), 500 μm.

Fig. 2. Paired SRS and H&E imaging of a frozen sectioned whole specimen with cancer cell infiltration

(A) The upper part of the specimen is basically white matter as shown by the strong lipid signals and the condensed myelin fibers, and the bottom part is gray matter with lower lipid signals. Red blood cells are shown in cyan color (marked by V). (B) Zoom-in images display clear white/gray matter margin (left) and single cell nuclei morphology, showing correlation between SRS and H&E (right). The presence of infiltrative cancer cells is confirmed by H&E stained image. Images were from frozen specimen B2 of case 15 (anaplastic oligodendroglioma, grade III). Scale bars: A, 500 μm; B, 100 μm.

Fig. 3. Quantification of cellularity with paired SRS and H&E imaging of frozen sectioned brain tissue

(A) SRS (left) and H&E stained (right) images of a whole specimen from a GBM case show hypercellularity. (B) Four zoom-in images in (A) demonstrate that the round-shape of the cell nuclei can be clearly visualized. (C) SRS (left) and H&E stained (right) images of a nonneoplastic tissue section show very low cell density (epilepsy). (D) Zoom-in images in (C) show clear single cell nuclei, and correlation between SRS and H&E images. Yellow dots in the zoom-in images demonstrate the cell counting procedure. Images in (A and B) were from frozen specimen #3 of case 22 (GBM) and images in (C and D) were from frozen specimen A1 of case 42 (epilepsy). Scale bar: (A, C), 500 μm; (B, D), 50 μm. (E) Linear correlation plot of cell counting from SRS and H&E images (correlation factor R² = 0.99). (F) Cell density tracing for non-neoplastic, glioma grade III, and GBM specimens. Data for non-neoplastic tissue include 41 (FOV) from the epilepsy case (case 42) and 69 FOV from the autopsy normal case A1. Data for glioma grade III include 15 FOV from case 39 and 27 FOV from case 15. Data for GBM include 29 FOV from case 22 and 6 FOV from case 9.

Fig. 4. SRS imaging of fresh human brain tumor tissue

(A) SRS image of the whole fresh specimen (~1 mm in thickness). (B) Four zoom-in images in (A) (yellow squares) show lower cell density, while the other four zoom-in images in (A) (magenta squares) show higher cell density (C), demonstrating tumor heterogeneity. Blood vessels with red blood cells are shown in cyan color. Single myelin fibers were visualized based on the strong lipid contrast with high sensitivity. Images were from fresh specimen BL5E-1 of case 44 (oligodendroglioma grade II). Scale bars: (A), 1 mm; (B, C), 100 μm.

Fig. 5. SRS and H&E stained imaging of blood vessels to characterize vascular proliferation in GBM

(A) SRS imaging of fresh brain tumor tissue shows blood vessels in blue with red blood cells (magenta). Cell nuclei of the endothelial cells of the blood vessels are clearly visualized based on the protein contrast. (B) Paired SRS and H&E imaging of the whole tissue section show vascular proliferation in a GBM case. (C) Zoom-in images of (B) show that the blood vessels can be visualized with high specificity by SRS imaging (marked by V). Images (A) were from fresh specimen 03#2 of case 46 (GBM), images (B and C) were from frozen specimen #2, case 22 (GBM). Scale bars: (A, C), 100 μm; (B), 500 μm.

Fig. 6. Paired SRS and H&E stained imaging of tissue from a GBM case shows extensive necrosis

(A) A specimen with extensive necrosis (below, marked by N) and a smaller area of viable tumor (upper, marked by T). (B) A specimen with a small viable tumor core surrounded by necrotic tissue. (C) A specimen with mixed distribution of viable tumor and necrosis. Zoom-in images in (A, C, E) are shown in (B, D F), respectively. Necrosis and viable tumor show very different morphological features and the boundary between these regions is resolved with sub-cellular resolution. All images were from case 9 (GBM). Scale bars: (A, C, E), 500 μm; (B, D, F), 100 μm.

Fig. 7. SRS imaging of massive collagen deposition in a fresh specimen from a recurrent/residual GBM surgical case

(A, C) Two representative large fields of view of the fresh human brain tumor tissue show the co-distribution of collagen and tumor cells. Both isolated single cells and cell clusters are observed. Zoom-in images of (A, C) are shown in (B, D), respectively. A lot of lipid droplets are found in the condensed cells region. Images were from fresh specimens of case 52. Scale bars: (A, C), 100 μm; (B, D), 50 μm.