Development of All-Fiber-Optic Scanning Non-Linear Endomicroscopy Technology and Potential Application for Preterm Birth Detection

An all-fiber-optic scanning endomicroscope has been developed [1-4], which is capable of performing highresolution nonlinear optical imaging including twophoton fluorescence and second harmonic generation (SHG) microscopy. The system is shown in Figure 1. The light source is a titanium-sapphire femtosecond laser. A customized double-clad fiber (DCF) with a single mode core and a large inner cladding is used for both delivering the excitation light (in the core) and collecting the generated nonlinear signal (primarily through the inner cladding). In order to maintain a narrow pulse width, the incident light is pre-chirped using a grating pair prior to being coupled into the DCF core using a lens. Beam scanning is achieved by threading the DCF through a four-quadrant tubular piezoelectric actuator (PZT). The four quadrants are driven electrically using sinusoidal waveforms with predetermined phase and amplitude relationships leading to an opening and closing spiral trajectory of the fiber tip. This leads to a spiral scan pattern of the focal spot on the sample. Light from the fiber is focused on the sample using an objective lens. The lens has a numerical aperture of 0.6 and is highly corrected to ensure a small focal spot leading to high spatial resolution. The PZT and the lens form the distal end of the probe which has a diameter under 2 mm and a rigid length of about 30 mm. The generated nonlinear signal is focused by the lens back into the fiber. The large inner core and the excellent achromaticity of the lens lead to a high collection efficiency, which is critical for high sensitivity and signal-to-noise ratio imaging. After exiting the fiber, the nonlinear signal is separated from the incident light using a dichroic mirror, bandpass filtered to isolate the signal of interest and directed to a photomultiplier tube for detection. Image reconstruction is done in real time on a computer at 2-3 frames/second. For SHG imaging, an excitation wavelength of 890 nm and bandpass filtering of 435-455 nm was used. About 40 mW average power was used for imaging. The above mentioned endomicroscopy technology literally miniaturizes a powerful bench-top laser scanning microscope to a flexible, fiber-optic endomicroscope with a very small footprint (i.e. an ~2 mm diameter) but without losing the essential functions and performance. The applications of the technology can be many. One of particular interest is preterm birth detection by assessing cervical collagen fiber morphology (through SHG imaging) during pregnancy, which is intimately related the mechanical rigidity of the cervix. Preterm birth is a significant problem with considerable infant mortality and neonatal care expenditure [5]. Recent research has shown that collagen morphology is correlated to the stage of pregnancy and therefore has the potential for early detection of preterm birth [6]. SHG is a nonlinear imaging technique that can be used to image biomolecular structures that lack centerosymmetry [7], such as collagen and actin. Collagen fiber structure, as revealed by SHG imaging, has been shown to correlate with the duration of gestation and the stage of pregnancy [8]. Our fiber-optic endomicroscopy platform can be used for in vivo imaging of collagen using SHG. For proof-of-concept, cervices from pregnant mice at varying stages of pregnancy were imaged ex vivo. Figures 2(a-c) show some representative cervical SHG images from a pregnant mouse at days 6, 12 and 18 of gestation. For comparison, Figures 2(d-f) show images of similar tissues, taken using a bench-top nonlinear laser scanning microscope equipped with a 20× 0.95 NA objective lens. In terms of sensitivity and resolution, the endomicroscope images are comparable to the microscope images while offering compatibility with a medical endoscopic examination and orders of magnitude savings in size and weight. From Figures 2(ac), clear changes in the organization and structure of the collagen can also be seen, which can potentially be used for detecting the stage of pregnancy and the possibility of preterm birth. The preliminary results suggest the potential role of the scanning fiber-optic SHG endomicroscopy for preterm birth detection.