Lasers in Surgery and Medicine Detection of Plaque Structure and Composition Using OCT Combined With Two-photon Luminescence (TPL) Imaging Tianyi Wang, PhD, 1 Austin McElroy, BS, 1 David Halaney, BS, 2,3 Deborah Vela, MD, 4 Edmund Fung, BS, 1 Shafat Hossain, BS, 1 Jennifer Phipps, PhD, 2 Bingqing Wang, PhD, 1 Biwei Yin, PhD, 1 Marc D. Feldman, MD, 2,3 and Thomas E. Milner, PhD 1 1 Department of Biomedical Engineering, University of Texas at Austin, 1 University Station C0800, Austin, Texas 2 Division of Cardiology, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, Texas 3 South Texas Veterans Health Care System, San Antonio, Texas 4 Texas Heart Institute, Houston, Texas Background and Objectives: Atherosclerosis and pla- que rupture leads to myocardial infarction and stroke. A novel hybrid optical coherence tomography (OCT) and two- photon luminescence (TPL) fiber-based imaging system was developed to characterize tissue constituents in the context of plaque morphology. Study Design/Materials and Methods: Ex vivo coro- nary arteries (34 regions of interest) from three human hearts with atherosclerotic plaques were examined by OCT–TPL imaging. Histological sections (4 mm in thick- ness) were stained with Oil Red O for lipid, Von Kossa for calcium, and Verhoeff–Masson Tri-Elastic for collagen/ elastin fibers and compared with imaging results. Results: Biochemical components in plaques including lipid, oxidized-LDL, and calcium, as well as a non-tissue component (metal) are distinguished by multi-channel TPL images with statistical significance (P < 0.001). TPL imaging provides complementary optical contrast to OCT (two-photon absorption/emission vs scattering). Merged OCT–TPL images demonstrate the distribution of lipid deposits in registration with detailed plaque surface profile. Conclusions: Results suggest that multi-channel TPL imaging can effectively identify lipid sub-types and different plaque components. Furthermore, fiber-based hybrid OCT–TPL imaging simultaneously detects plaque structure and composition, improving the efficacy of vulnerable plaque detection and characterization. Lasers Surg. Med. © 2015 Wiley Periodicals, Inc. Key words: atherosclerotic plaque; lipid; oxidized-LDL; calcium; collagen/elastin fiber; hybrid imaging; optical coherence tomography; two-photon luminescence ima- ging; photonic crystal fiber INTRODUCTION Despite continuing progress of optical imaging techni- ques in clinical diagnostics, therapeutics and intervention guidance [1–5], new imaging approaches are needed for improved efficacy of disease detection. In this study, we demonstrate a fiber-based hybrid optical coherence tomography and two-photon luminescence (OCT–TPL) imaging system to characterize atherosclerotic plaques in ex vivo human coronary arteries. The imaging system has advantages over OCT or TPL imaging alone, possessing two complementary types of optical contrast (i.e., two- photon absorption/emission and scattering). Further development of OCT–TPL imaging may provide cardiolo- gists a diagnostic imaging tool that can simultaneously record co-registered images of plaque structure and biochemical composition, and improve the accuracy of vulnerable plaque detection. The principal pathologic features of atherosclerotic plaques prone to rupture are well described. Davies et al noted that with the reconstruction of serial histological sections in individuals suffering from acute myocardial infarctions associated with death, a rupture or fissuring of “vulnerable plaque” was evident [6]. Vulnerable plaques, recently defined by Virmani [7] using a more descriptive term “thin-cap fibroatheroma (TCFA)”, were further characterized as having a thin fibrous cap (typically less than 65 mm thick) overlaying a lipid core, increased infiltration of macrophages, and decreased smooth muscle cells compared to stable plaques [8–10]. Many of the cellular/molecular events that lead to rupture of TCFAs, thrombus formation and consequent acute myocardial infarction are now understood and being utilized to develop novel imaging approaches and therapeutic interventions. Several features during atherogenesis have been identified that contribute to mechanical instability and increased Contract grant sponsor: ASLMS Research Grant; Contract grant sponsor: Veterans Health Administration Merit Grant; Contract grant number: I01 BX000397; Contract grant sponsor: American Heart Association Grant; Contract grant number: 13POST17080074; Contract grant sponsor: Clayton Foundation for Biomedical Research; Contract grant sponsor: University of Texas.  Correspondence to: Tianyi Wang, PhD, Department of Biomedical Engineering, 1 University Station C0800, Austin, Texas 78712. E-mail: Tianyi.Wang@utexas.edu Accepted 5 April 2015 Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/lsm.22366 ß 2015 Wiley Periodicals, Inc.