Stephen A Boppart Mark E Brezinski and James G Fujimoto 1 Introduction

Optical coherence tomography (OCT) is an attractive imaging technique for developmental biology because it permits the imaging of tissue microstructure in situ, yielding micron-scale image resolution without the need for excision of a specimen and tissue processing. OCT enables repeated imaging studies to be performed on the same specimen in order to track developmental changes. OCT is analogous to ultrasound B mode imaging except that it uses low-coherence light rather than sound and performs cross-sectional imaging by measuring the backscattered intensity of light from structures in tissue (1). The principles of OCT imaging are shown schematically in Fig. 1. The OCT image is a gray-scale or false-color two-dimensional (2-D) representation of backscattered light intensity in a cross-sectional plane. The OCT image represents the differential backscattering contrast between different tissue types on a micron scale. Because OCT performs imaging using light, it has a one- to two-order-of-magnitude higher spatial resolution than ultrasound and does not require specimen contact.

OCT was originally developed and demonstrated in ophthalmology for high-resolution tomographic imaging of the retina and anterior eye (2-4). Because the eye is transparent and is easily optically accessible, it is well suited for diagnostic OCT imaging. OCT is promising for the diagnosis of retinal disease because it can provide images of retinal pathology with 10-^m resolution, almost one order of magnitude higher than previously possible using ultrasound. Clinical studies have been performed to assess the application of OCT for a number of macular diseases (3,4). OCT is especially promising for the diagnosis and monitoring of glaucoma and macular edema associated with diabetic retinopathy because it permits the quantitative measurement of changes in the retinal or retinal-nerve fiber layer thickness. Because morphological changes often occur before the onset of physical symptoms, OCT can provide a powerful approach for the early detection of these diseases.

Recently, OCT has been applied for imaging in a wide range of nontransparent tissues (5-9). In tissues other than the eye, the imaging depth is limited by optical attenuation resulting from scattering and absorption. Ophthalmic imaging is typically performed at 800-nm wavelengths. However, because optical scattering decreases with

From: Methods in Molecular Biology, Vol. 135: Developmental Biology Protocols, Vol. I Edited by: R. S. Tuan and C. W. Lo © Humana Press Inc., Totowa, NJ

Fig. 1. OCT imaging is performed by directing an optical beam at the object to be imaged, and the echo delay of backscattered light is measured.

increasing wavelength, OCT imaging in nontransparent tissues is possible using 1.3 ^m or longer wavelengths. In most tissues, imaging depths of 2-3 mm can be achieved using a system detection sensitivity of 100-110 dB. OCT has been applied to image arterial pathology in vitro and has been shown to differentiate plaque morphology with superior resolution to ultrasound (10-12). Imaging studies have also been performed to investigate applications in gastroenterology, urology, and neurosurgery (13-15). Highresolution OCT using short-coherence-length, short-pulsed light sources has also been demonstrated and axial resolutions of <5 ^m achieved (16,17). High-speed OCT at image acquisition rates of 4-8 frames/s for a 250- to 500-square pixel images has been achieved (18,19). OCT has been extended to perform Doppler imaging of blood flow and birefringence imaging to investigate laser intervention (20-22). Different imaging delivery systems, including transverse imaging catheter/endoscopes and forward-imaging devices, have been developed to enable internal body OCT imaging (23,24). Most recently, OCT has been combined with catheter/endoscope-based delivery to perform in vivo imaging in animal models (25).

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