Differential Diagnosis of Lung Carcinoma With Coherent Anti-Stokes Raman Scattering Imaging Liang Gao, PhD; Zhiyong Wang, PhD; Fuhai Li, PhD; Ahmad A. Hammoudi, MS; Michael J. Thrall, MD; Philip T. Cagle, MD; Stephen T. C. Wong, PhD, PE  Aimed at bridging imaging technology development with cancer diagnosis, this paper first presents the prevailing challenges of lung cancer detection and diagnosis, with an emphasis on imaging techniques. It then elaborates on the working principle of coherent anti- Stokes Raman scattering microscopy, along with a de- scription of pathologic applications to show the effective- ness and potential of this novel technology for lung cancer diagnosis. As a nonlinear optical technique probing intrinsic molecular vibrations, coherent anti-Stokes Raman scattering microscopy offers an unparalleled, label-free strategy for clinical cancer diagnosis and allows differen- tial diagnosis of fresh specimens based on cell morphology information and patterns, without any histology staining. This powerful feature promises a higher biopsy yield for early cancer detection by incorporating a real-time imaging feed with a biopsy needle. In addition, molecu- larly targeted therapies would also benefit from early access to surgical specimen with high accuracy but minimum tissue consumption, therefore potentially saving specimens for follow-up diagnostic tests. Finally, we also introduce the potential of a coherent anti-Stokes Raman scattering–based endoscopy system to support intraoper- ative applications at the cellular level. (Arch Pathol Lab Med. 2012;136:1502–1510; doi: 10.5858/arpa.2012-0238-SA) L ung carcinoma is the most prevalent type of cancer in the world and is responsible for more deaths than other types of cancer. 1 When symptoms occur, the disease often has already progressed to an advanced stage, which is the major cause of its low survival rate. 2,3 Five-year survival rates after diagnosis are between 8% and 12% in Europe 3,4 and less than 15% in North America. 2 Early detection of lung cancer has attracted major research interest because it dramatically increases the survival rate. 5,6 Conventional detection strategies like sputum cytology 7–9 and x-ray 10–12 confront significant limitations because of their low detection rates. 13,14 As a result, new diagnostic technologies have been actively developed in the past few decades; some of them have been used in clinical applications. Low-dose spiral computed tomography has been shown to be capable of detecting lung nodules at an earlier stage than conventional chest x-ray. 15,16 Nodules as small as 2 to 3 mm 7 can be detected with low-dose spiral computed tomography, such that many lung cancers can be detected at stage 1. However, the low specificity of this method leads to a large number of false-positive readings and unneces- sary follow-up evaluations in a significant number of tested patients. 17 To further assess the pulmonary nodules (.7–8 mm) detected by computed tomography, positron emission tomography has been deployed, 18,19 particularly in charac- terizing the staging and possible metastasis of cancer. 20,21 Nevertheless, this technique suffers from low spatial resolution 22 and poor susceptibility to respiratory motion. 23 As a result, a tissue biopsy (normally fine-needle aspiration) is always included as a follow-up test after the detection of a nodule. The problem encountered by fine-needle aspiration, however, is the difficulty of performing biopsy on nodules smaller than 10 mm, 17 making the diagnosis of small lung lesions a continuing difficulty. Clinical problems in early lung cancer detection have driven the development of bronchoscopies. Whereas conventional white-light bronchoscopy is based on the detection of alterations in tissue surface structure, autofluo- rescence bronchoscopy aims at exploiting the spectral difference between normal and precancerous/early cancer- ous tissues. 24 The only approved clinical device (Xillix Technologies, Richmond, British Columbia, Canada) takes advantage of the fact that cancerous lesions possess a higher level of backscattered red light than normal tissue when excited with a violet laser. Size and specificity are the major limiting factors of this technique because smaller–fiber-optic instruments (,1 mm) are needed for diagnosis of peripheral lesions. 17 As an emerging label-free technique, optical coherent tomography formulates optical contrasts using the interfer- ence of 2 temporally incoherent lights, with a resolution of 2 Accepted for publication May 18, 2012. From the Department of Systems Medicine and Bioengineering (Drs Gao, Wang, Li, and Wong and Mr Hammoudi) and the NCI- ICBP Center for Modeling Cancer Development (Drs Li and Wong), The Methodist Hospital Research Institute, and the Department of Pathology and Genomic Medicine, The Methodist Hospital (Drs Thrall, Cagle, and Wong), Weill Cornell Medical College of Cornell University, Houston, Texas; Chroma Technology Corporation, Bellows Falls, Vermont (Dr Gao); and the Department of Electrical and Computer Engineering, Rice University, Houston, Texas (Mr Hammoudi and Dr Wong). The authors have no relevant financial interest in the products or companies described in this article. Presented at the Houston Lung Symposium; April 28–29, 2012; Houston, Texas. Reprints: Stephen T. C. Wong, PhD, PE, Department of Systems Medicine and Bioengineering, The Methodist Hospital, 6565 Fannin St, Houston, TX 77030 (e-mail: STWong@tmhs.org). 1502 Arch Pathol Lab Med—Vol 136, December 2012 Diagnosis of Lung Carcinoma With CARS—Gao et al