High-content stimulated Raman histology of human breast cancer

Abstract

Histological examination is crucial for cancer diagnosis, however, the labor-intensive sample preparation involved in the histology impedes the speed of diagnosis. Recently developed two-color stimulated Raman histology could bypass the complex tissue processing to generates result close to hematoxylin and eosin staining, which is one of the golden standards in cancer histology. Yet, the underlying chemical features are not revealed in two-color stimulated Raman histology, compromising the effectiveness of prognostic stratification. Here, we present a high-content stimulated Raman histology (HC-SRH) platform that provides both morphological and chemical information for cancer diagnosis based on un-stained breast tissues. Methods: By utilizing both hyperspectral SRS imaging in the C-H vibration window and sparsity-penalized unmixing of overlapped spectral profiles, HC-SRH enabled high-content chemical mapping of saturated lipids, unsaturated lipids, cellular protein, extracellular matrix (ECM), and water. Spectral selective sampling was further implemented to boost the speed of HC-SRH. To show the potential for clinical use, HC-SRH using a compact fiber laser-based stimulated Raman microscope was demonstrated. Harnessing the wide and rapid tuning capability of the fiber laser, both C-H and fingerprint vibration windows were accessed. Results: HC-SRH successfully mapped unsaturated lipids, cellular protein, extracellular matrix, saturated lipid, and water in breast tissue. With these five chemical maps, HC-SRH provided distinct contrast for tissue components including duct, stroma, fat cell, necrosis, and vessel. With selective spectral sampling, the speed of HC-SRH was improved by one order of magnitude. The fiber-laser-based HC-SRH produced the same image quality in the C-H window as the state-of-the-art solid laser. In the fingerprint window, nucleic acid and solid-state ester contrast was demonstrated. Conclusions: HC-SRH provides both morphological and chemical information of tissue in a label-free manner. The chemical information detected is beyond the reach of traditional hematoxylin and eosin staining and heralds the potential of HC-SRH for biomarker discovery.

Keywords: breast cancer; fiber laser; high-content chemical imaging; label-free histology; stimulated Raman histology.

Figure 1

System schematic. (A) A state-of-the-art SRS imaging system with a solid-state laser. (B) A compact fiber laser-based SRS imaging system. OPO: optical parametric oscillator. AOM: acousto-optic modulator. PD: Photodiode. VGA: Variable gain amplifier. PID: proportional-integral-derivative controller. BS: Beam Splitter

Figure 2

HC-SRH of human breast cancer tissue slice. (A) Merged concentration map for a breast cancer tissue sample. Yellow for unsaturated lipids, red for cellular protein, blue for ECM, cyan for saturated lipids, and grey for water. (B) Separate concentration maps for the 5 chemical components and the reference spectra corresponding to (A). (C) H&E result of a neighboring section to the tissue section used in (A). (D) Zoom-in view of the white box in (a) and SRS spectra of the five selected pixels as numbered. (E) Comparison of HC-SRH and SHG in mapping ECM in a cancer-adjacent vessel. Scale bar: 50 µm.

Figure 3

High-speed HC-SRH via spectral selective sampling. Scale bar: 50 µm. SSIM: structural similarity. PSNR: peak signal-to-noise ratio.

Figure 4

Breast tissue features revealed by HC-SRH. (A-D) HC-SRH of breast lesion tissue with 5-channel selective spectral sampling. A: typical ductal carcinoma in-situ (DCIS); B: mixture of usual ductal hyperplasia (UDH), normal duct, and invasive ductal carcinoma (IDC); C: IDC and invasive lobular carcinoma mixture (ILC); D: Chemotherapy lesion and some ILC residue. (E-H) H&E of neighboring tissue section corresponding to (A-D). (I-L) Zoom-in of ROIs 3,4,6,9. Color representation: Yellow: Unsaturated lipid, Red: Cell protein, Blue: Extracellular matrix, Cyan: Saturated fat. Scale bar: 200 µm.

Figure 5

HC-SRH with a compact fiber laser. (A.) Hyperspectral HC-SRH (61 spectral channel) acquired by the FOPO-laser-based SRS system. (B) Selective sampled HC-SRH (5 spectral channel) acquired by the FOPO-laser-based SRS system. The spectral channel position is the same with Figure 3. (C) Selectively sampled HC-SRH (5 spectral channel) acquired in solid state OPO-based SRS system. Color representation: Yellow: Unsaturated lipid, Red: Cell protein, Blue: Extracellular matrix, Cyan: Saturated fat. Scale bar: 50 µm.

Figure 6

Fingerprint SRS imaging of breast cancer tissue. (A) C-H HC-SRH result. Color representation: Yellow: Unsaturated lipid, Red: Cell protein, Blue: Extracellular matrix, Cyan: Saturated fat. (B) Fingerprint SRS result at representative spectral position in the same FOV of (A). (C) Multi-window SRS spectra of the 4 selected ROIs in (A). Scale bar: 10 µm.