Development of Molecular Contrast-enhanced Imaging for Optical Coherence Tomography

Biological imaging techniques that are able to detect a contrast-enhanced signal from the target molecules have been widely applied to various techniques in the imaging field. The complex biological environment provides numerous and more efficient pathways along which the chromophores (light absorber) may release its energy. This energy can provide not only morphological information, but also specific molecular information such as a biochemical map of a sample. All diseases correlate with both morphological and biochemical changes. Optical coherence tomography (OCT) system is one of the biological imaging techniques. OCT has widely been applied to many medical/clinical fields, giving benefit from a penetration depth of a few millimeters while maintaining a spatial resolution on the order of a micron. Unfortunately, OCT lacks the straightforward functional molecular imaging extensions available for other technologies, e.g. confocal fluorescence microscopy and fluorescence diffuse optical tomography. This is largely because incoherent processes such as fluorescence emission and Raman scattering are not readily detectable with low coherence interferometry that is the central technique that underlies all OCT systems. Despite a drawback of molecular imaging with OCT, it is highly desirable to measure not only morphological, but also molecular information from either endogenous or exogenous molecules. In order to overcome the limitation of molecular contrast imaging for OCT, our group has been researched the hybrid OCT imaging technique and a new exogenous contrast agent. Our contrast-enhanced imaging technique integrates OCT with a well-researched and well-established technique: two-colored pump-probe absorption spectroscopy. Our novel imaging technique is called Pump-Probe OCT (PPOCT). Based upon current successful results, molecular imaging with OCT potentially gives us the ability to identify pathologies. In order to expand the capacity of PPOCT, this dissertation focuses on development of molecular contrast-enhanced imaging for optical coherence tomography (OCT). In the first phase of the research, we developed and optimized for sensitivity a two-color ground state recovery Pump-Probe Optical Coherence Tomography (gsrPPOCT) system and signal algorithm to measure the contrast-enhanced signal of endogenous and exogenous contrast agents such as Hemoglobin (Hb) and Methylene blue (MB) from in vivo samples. Depending on the absorption peak of a target molecule, the pump light sources for PPOCT used 532nm Q-switched laser or 663nm diode laser. Based on different experimental application, Ti:sapp or SLD of 830nm center wavelength were utilized. The PPCOT system was firstly used to image Hb of in vivo vasulature in a Xenopus laevis as the endogenous contrast agent and a larval stage zebrafish using MB as the exogenous contrast agent via transient changes in light absorption. Their morphological in addition to molecular specific information from a live animal was described. The incorporation of a pump laser in an otherwise typical spectrometer based OCT system is sufficient to enable molecular imaging with PPOCT. In the second phase of this research, based on endoscopic molecular contrast-enhanced applications for OCT, we invented an ultra-wideband lensless fiber optic rotary joint based on co-aligning two optical fibers has excellent performance (~0.38 dB insertion loss). The developed rotary joint can cover a wavelength range of at least 355- 1360 nm with single mode, multimode, and double clad fibers with rotational velocities up to 8800 rpm (146 Hz). In the third phase of this research, we developed and manufactured a microencapsulated methylene blue (MB) contrast agent for PPOCT. The poly lactic coglycolic acid (PLGA) microspheres loaded with MB offer several advantages over bare MB. The microsphere encapsulation improves the PPOCT signal both by enhancing the scattering and preventing the reduction of MB to leucomethylene blue. The surface of the microsphere can readily be functionalized to enable active targeting of the contrast agent without modifying the excited state dynamics of MB that enable PPOCT imaging. Both MB and PLGA are used clinically. PLGA is FDA approved and used in drug delivery and tissue engineering applications. 2.5 µm diameter microspheres were synthesized with an inner core containing 0.01% (w/v) aqueous MB. As an initial demonstration the MB microspheres were imaged in a 100 µm diameter capillary tube submerged in a 1% intralipid emulsion. By varying the oxygen concentration both 0% and 21%, we observed he lifetime of excited triple state using time-resolved Pump-Probe spectroscopy and also the relative phase shift between the pump and probe is a reliable indicator of the oxygen concentration. Furthermore, these results are in good agreement with our theoretical predictions. This development opens up the possibility of using MB for 3-D oxygen sensing with PPOCT