Multimodal non-linear optical imaging for label-free differentiation of lung cancerous lesions from normal and desmoplastic tissues

Abstract

Lung carcinoma is the leading cause of cancer-related death in the United States, and non-small cell carcinoma accounts for 85% of all lung cancer cases. One major characteristic of non-small cell carcinoma is the appearance of desmoplasia and deposition of dense extracellular collagen around the tumor. The desmoplastic response provides a radiologic target but may impair sampling during traditional image-guided needle biopsy and is difficult to differentiate from normal tissues using single label free imaging modality; for translational purposes, label-free techniques provide a more promising route to clinics. We thus investigated the potential of using multimodal, label free optical microscopy that incorporates Coherent Anti-Stokes Raman Scattering (CARS), Two-Photon Excited AutoFluorescence (TPEAF), and Second Harmonic Generation (SHG) techniques for differentiating lung cancer from normal and desmoplastic tissues. Lung tissue samples from patients were imaged using CARS, TPEAF, and SHG for comparison and showed that the combination of the three non-linear optics techniques is essential for attaining reliable differentiation. These images also illustrated good pathological correlation with hematoxylin and eosin (H&E) stained sections from the same tissue samples. Automated image analysis algorithms were developed for quantitative segmentation and feature extraction to enable lung tissue differentiation. Our results indicate that coupled with automated morphology analysis, the proposed tri-modal nonlinear optical imaging technique potentially offers a powerful translational strategy to differentiate cancer lesions reliably from surrounding non-tumor and desmoplastic tissues.

Keywords: (170.2520) Fluorescence microscopy; (170.3880) Medical and biological imaging; (170.4580) Optical diagnostics for medicine; (190.1900) Diagnostic applications of nonlinear optics; (270.4180) Multiphoton processes; (300.6230) Spectroscopy, coherent anti-Stokes Raman scattering.

Fig. 1

TPEAF, SHG, CARS, and H&E images of normal lung tissue. (A) TPEAF image. (B) SHG image. (C) CARS image. (D) Superimposed TPEAF and SHG image. (E) Superimposed TPEAF, SHG, and CARS images. (F) H&E image of the same lung tissue but at a different location. Green indicates the TPEAF of elastin fiber in the range of 500-600 nm. Blue indicates the SHG signal from collagen fiber at 408.5 nm. CARS image (grey scale) shows signals from cells and fibrous structures of elastin and collagen. Yellow arrow and blue arrow point to a cell with a central dark nucleus and fibrous protein, respectively. Scale bar: 50 µm.

Fig. 2

TPEAF, SHG, CARS, and H&E images of adenocarcinoma tissue. (A) TPEAF image of adenocarcinoma tissue. (B) SHG image of adenocarcinoma tissue. (C) CARS image of adenocarcinoma tissue. (D) Superimposed TPEAF and SHG image of adenocarcinoma tissue. (E) Superimposed TPEAF, SHG, and CARS images of adenocarcinoma tissue. (F) H&E image of the same adenocarcinoma tissue. Green indicates the TPEAF of elastin fiber in the range of 500-600 nm. Blue indicates the SHG signal from collagen fiber at 408.5 nm. CARS image (grey scale) shows signals from cells and fibrous structures of elastin and collagen. Yellow arrow and blue arrow point to a cell with a central dark nucleus and fibrous protein, respectively. Scale bar: 50 µm.

Fig. 3

TPEAF, SHG, CARS, and H&E images of squamous cell carcinoma tissue. (A) TPEAF image of squamous cell carcinoma tissue. (B) SHG image of squamous cell carcinoma tissue. (C) CARS image of squamous cell carcinoma tissue. (D) Superimposed TPEAF and SHG image of squamous cell carcinoma tissue. (E) Superimposed TPEAF, SHG, and CARS images of squamous cell carcinoma tissue. (F) H&E image of the same squamous cell carcinoma tissue. Green indicates the TPEAF of elastin fiber in the range of 500-600 nm. Blue indicates the SHG signal from collagen fiber at 408.5 nm. CARS image (grey scale) shows signals from cells and fibrous structures of elastin and collagen. Yellow arrow and blue arrow point to a cell with a central dark nucleus and fibrous protein, respectively. Scale bar: 50 µm.

Fig. 4

TPEAF, SHG, CARS, and H&E images of desmoplastic edge. (A) TPEAF image. (B) SHG image. (C) CARS image. (D) Superimposed TPEAF and SHG image. (E) Superimposed TPEAF, SHG, and CARS images. (F) H&E image of the same lung tissue. (G) A low magnification image that shows the position of the imaged area (Black square). Green indicates the TPEAF of elastin fibers in the range of 500-600 nm. Blue indicates the SHG signal from collagen fibers at 408.5 nm. CARS image (grey scale) shows signals from a cell with a central dark nucleus, a lipid-rich focus, and fibrous structures of elastin and collagen. Yellow arrow and blue arrow point to a cell nucleus and fiber protein, respectively.

Fig. 5

(A) Segmented results of elastin fibers in an example TPEAF image of normal lung tissue. (B) Segmented results of collagen fibers in an SHG image of normal lung tissue.

Fig. 6

Comparison of normal, desmoplastic and tumor tissues in multimodal images. (A) Collagen fibers in SHG images. (B) Elastin fibers in TPEAF images. A total of six images of normal tissues, 13 images of desmoplastic edge tissues, and 33 images of tumorous tissues are analyzed in this study.