Ординатура / Офтальмология / Английские материалы / Handbook of Optical Coherence Tomography_Bouma, Tearney_2002

the three different materials, PMMA, acrylic, and silicone, which are the most common materials in use today in cataract surgery. The capsular bag could be identified but in no case could the intraocular lens itself be visualized by the OCT. The reason for this phenomenon is probably the homogeneous and extremely translucent material of the lenses and their smooth surface. Therefore only a punctate specular reflection could be detected, and no structure could be visualized within the OCT image.

18.2.8 Potential and Limitations of OCT in the Anterior Segment

Imaging in ophthalmology has considerably improved over recent years, but only selected techniques allow high resolution imaging of the anterior segment [15,17]. The first clinical experiences with the slitlamp-adapted OCT system, which was developed for routine clinical examinations of the anterior and posterior segments, are promising. OCT measurements performed with this system deliver high resolution cross-sectional images of intraocular structures using the familiar slitlamp examination technique, thus extending the use of OCT to the anterior globe. It allows a quick change between anterior and posterior segment examinations and provides exact and rapid biometric analyses of structures and dimensions in the anterior globe without direct contact or immersion techniques. Important clinical measurements include corneal thickness, corneal surface profile, corneal refraction, iris thickness, anterior chamber angle, lens thickness, and thickness of secondary cataract formation. A drawback is that direct morphometric analysis is possible only for axial measurements. Off-axis measurements need further correction. For this correction, knowledge of the profile of the ocular structures anteriorly to the structure of interest is required.

18.3PERSPECTIVES

Our preliminary results suggest a wide range of potential clinical and scientific applications for the slitlamp-adapted OCT system presented here for examination of the anterior segment of the eye. Further technical improvements will shorten the acquisition time and reduce motion artifacts. Optimization of the coherence signal demodulation and image processing can improve the sensitivity and contrast of the OCT images. Increasing the length of the lateral scans will provide a better analysis of the complete anterior segment. Simultaneous documentation of the scanned area in related color photographs will further improve the reproducibility of measurements and orientation within the images.

Further studies will have to investigate the infrared OCT technique, which has the potential to replace ultrasound biomicroscopy. Current technical refinements and the slitlamp adaptation of OCT will improve imaging quality for the examination of the anterior segment of the eye.

REFERENCES

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2.Hee MR, Puliafito CA, Duker JS, Reichel E, Coker JG, Wilkins JR, Schuman JS, Swanson EA, Fujimoto JG. Topography of diabetic macular edema with optical coherence tomography. Ophthalmology 105:360–370, 1998.

3.Puliafito CA, Hee MR, Schuman JS, Fujimoto JG. Optical Coherence Tomography of Ocular Diseases. Thorofare, NJ: Slack Inc, 1996.

4.Rutledge BK, Puliafito CA, Duker JS, Hee MR, Cox MS. Optical coherence tomography of macular lesions associated with optic nerve head pits. Ophthalmology 103:104– 105, 1996.

5.Schuman JS, Hee MR, Puliafito CA, Wong C, Pedut-Kloizman T, Lin CP, Hertzmark E, Izatt JA, Swanson EA, Fujimoto JG. Quantification of nerve fiber layer thickness in normal and glaucomatous eyes using optical coherence tomography. Arch Ophthalmol 113:586–596, 1995.

6.Toth CA, Birngruber R, Boppart SA, Hee MR, Fujimoto JG, DiCarlo CD, Swanson EA, Cain CP, Narayan DG, Noojin GD, Roach WP. Argon laser retinal lesions evaluated in vivo by optical coherence tomography. Am J Ophthalmol 123:188–198, 1997.

7.Wilkins JR, Puliafito CA, Hee MR, Duker JS, Reichel E, Coker JG, Schuman JS, Swanson EA, Fujimoto JG. Characterization of epiretinal membranes using optical coherence tomography. Ophthalmology 103:2142–2151, 1996.

8.Izatt JA, Hee MR, Swanson EA, Lin CP, Huang D, Schuman JS, Puliafito CA, Fujimoto JG. Micrometer-scale resolution imaging of the anterior eye in vivo with optical coherence tomography. Arch Ophthalmol 112:1584–1589, 1994.

9.DiCarlo CD, Boppart S, Gagliano DA, Amnotte R, Smith AB, Hammer DX, Cox AB, Hee MR, Fujimoto J, Swanson E, Roach WP. A new noninvasive imaging technique for cataract evaluation in the rheus monkey. In: Proceedings of Lasers in Surgery: Advanced Characterization, Therapeutics, and Systems V, 1995. SPIE 2395: 636–643.

10.Koop N, Brinkman R, Lankenau E, Flache S, Engelhardt R, Birngruber R. Optische Koha¨renztomographie der Kornea and des vorderen Augenabschnitts. Ophthalmologe 94:481–486, 1997.

11.Hoerauf H, Wirbelauer C, Scholz C, Engelhardt R, Koch P, Laqua H, Birngruber R. Slitlamp-adapted optical coherence tomography (OCT) of the anterior segment. Graefe’s Arch Clin Exp Ophthalmol 1999.

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13.Huebscher HJ, Genth U, Seiler T. Determination of excimer laser ablation rate of the human cornea using in vivo Scheimpflug videography. Invest Ophthalmol Vis Sci 1996:37–42.

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resonance imaging (MRI) and ultrasound. High image resolutions (2–10 m) with 2– 3 mm imaging penetration depths in scattering tissue permit the microscopic visualization of dynamic changes that occur during all stages of embryonic development. High speed OCT imaging enables functional assessment of developmental changes such as those within the cardiovascular system. By imaging developing specimens in vivo at near-histological resolutions, fewer animals will have to be killed at single time points for longitudinal studies. OCT permits the longitudinal tracking of morphological and functional development in single specimens. Thus, OCT promises to become a powerful and unique investigative tool for developmental biology.

19.2DEVELOPMENTAL BIOLOGY ANIMAL MODELS

The field of developmental biology uses several common animal models ranging from prokaryotic bacteria and eukaryotic yeast to increasingly more advanced nematodes (worm, Caenorhabditis elegans), fish (zebra fish, Brachydanio rerio), insects (fruit fly, Drosophila melanogaster), amphibians (African frog, Xenopus laevis), birds (chicken, Gallus domesticus), and small mammals (mouse, Mus musculus; rat, Rattus norvegicus). The lower species on the evolutionary tree are preferred for their ease of care and handling and their rapid reproductive cycles. Mice and rats are preferred for their close homology to humans, although even the single-cell yeast has a significant degree of homology. The advancements in molecular biological techniques have permitted researchers to site-specifically modify the genomes of these animal models. By modifying the genome and observing how mutations are expressed within the developing organism, locations and functions of specific genes can be determined. OCT can be applied to developmental biology to observe the expression of genes in vivo.

The high resolution and high speed imaging capabilities of OCT make it well suited for imaging the small animal models used in developmental biology. These animal models are also interesting for demonstrating OCT because of their small size, ease of care and handling, variations in optical transparency, intact in vivo functioning organ systems, and high cellular mitotic rates. Common developmental animal models, namely amphibians and fish, are used in a series of experiments that not only demonstrate the capabilities of OCT as an imaging modality for biological microscopy and developmental biology but also demonstrate the principles of high resolution OCT imaging.

19.3IDENTIFICATION OF TISSUE MORPHOLOGY

Improved imaging of morphological changes has the potential for offering new insight into the complex process of embryonic development. Imaging embryonic morphology that results from cellular differentiation is important for the understanding of genetic expression, regulation and control. Several well-recognized imaging technologies are currently used to provide structural information about microscopic specimens. These include magnetic resonance imaging, computer tomography, ultrasound, and confocal microscopy. High resolution magnetic resonance imaging has been used to image the mouse embryonic cardiovascular system [9] as well as to produce in vivo cross-sectional images of early Xenopus laevis development [10,11] with resolutions of 12 m. Because the static and gradient magnetic fields

required to obtain these resolutions are orders of magnitude greater than those found in most clinical systems, this modality represents a technically challenging option that requires considerable skill from its operator in order to achieve high resolution images. High resolution computed tomographic imaging of fixed insect specimens revealed internal microstructure with 8–12 m resolution yet required an elaborate microfocusing instrument and image reconstruction algorithms [12]. Ultrasound backscatter microscopy using high frequencies (40–100 MHz) is capable of 50 m resolutions to depths of 4–5 mm and has been applied to the analysis of early embryonic development in the mouse [13]. To effectively image with ultrasound, probes require contact with the tissue.

The invention of the confocal microscope [14] and laser-scanning confocal microscopy has advanced the understanding of biological systems and their development largely due to the ability to selectively visualize biological specimens, cells, and subcellular constituents [15]. Transverse resolutions of 0:5 m with 1 m optical sections are possible [16]. Although confocal microscopy is superb for optically sectioning a specimen, imaging depths are limited to less than 500 m in nontransparent tissue [17]. Recent advances in confocal microscopy have successfully shown that in vivo confocal imaging is possible. Examples include the imaging of calcium dynamics in sea urchin eggs [18] as well as Xenopus oocytes [19] during fertilization. Obtaining in vivo images is difficult, however, primarily due to the toxicity of the products that are released when the fluorophores are excited by the incident laser radiation. This limitation, as well as limited imaging penetration, prevents biologists from imaging structures over time, at later developmental stages, and in highly scattering, optically opaque specimens. Currently, internal morphological changes occurring in later stages can be studied only with histological preparations at discrete time points.

An in vivo means of imaging morphology is frequently needed to help identify the expressions of genes. Furthermore, observing and tracking morphological changes throughout development is useful for characterizing all aspects of genetic expression. OCT can perform high resolution, non-contact, cross-sectional tomographic imaging in vivo with the potential to analyze the morphological changes in both semitransparent and highly scattering specimens during normal and abnormal development. Optical coherence tomographic imaging was performed on several of the commonly used animal models in order to establish baselines and demonstrate the domains of application for this technology [5–8]. To verify image representation of morphology, OCT images were correlated with standard histological observations of the specimen, the current gold standard for identifying morphological features during development.

Optical coherence tomography has the ability to image specimens that are opaque to visible light because it uses wavelengths of light in the near-infrared region. Although most tissues appear opaque under visible light, they are relatively nonabsorbing in the near-infrared. Imaging depth using near-infrared light is limited by attenuation from optical scattering rather than absorption. All of the OCT imaging described in this chapter employed low coherence light sources with center wavelengths around 1300 nm. In general, a superluminescent diode was used for imaging stationary tissue structures. However, to achieve a sufficient signal-to-noise ratio (SNR), acquisition times were slow, ranging from 10 to 30 s depending on the image size. Because the SNR is proportional to the incident optical power, faster

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OCT image acquisition requires higher optical powers. A solid-state Kerr lens modelocked Cr4þ:forsterite laser was used for high speed OCT imaging [20]. For both of these low coherence light sources, OCT imaging depths up to 3 mm were possible. Although this imaging depth is not as great as that of ultrasound, it is sufficient for imaging many anatomical features of interest in most developing embryos. Throughout this chapter, OCT imaging parameters specific to each study will be described.

The first investigation of OCT in developmental biology examined the morphology of developing tadpoles [5]. For these studies, a superluminescent diode was used with a free-space longitudinal spatial resolution of 16 m (as determined by the 50 nm optical bandwidth of the low coherence light source). The transverse resolution was set to be 30 m (as determined by the spot size of the light beam). Within the specimen, a longitudinal resolution of 12 m was determined by dividing the free-space resolution by the measured average index of refraction of the specimen [21], n ¼ 1:35.

The transverse resolution for the following OCT images was 30 m with a corresponding confocal parameter of 1.1 mm. The SNR was 109 dB. The fact that OCT uses low coherence light and detects light at selected echo time delays greatly discriminates against the detection of light that is multiply scattered. Whereas confocal microscopy discriminates against unwanted light by using spatial filtering, photons can be multiply scattered into the spatial mode that is detected and degrade image quality [22].

Imaging studies were performed on several standard biological animal models commonly employed in developmental biology investigations. The animals used in these research studies were cared for and maintained under the established and approved protocols of the Committee on Animal Care, Massachusetts Institute of Technology, Cambridge, MA. OCT imaging was performed in Rana pipiens tadpoles (in vitro), Brachydanio rerio embryos and eggs (in vivo), and Xenopus laevis tadpoles (in vivo). Tadpoles were anesthetized by immersion in 0.5% Tricaine until they no longer responded to touch. Specimens were oriented for imaging with the optical beam incident from either the dorsal or ventral sides. After imaging, specimens for histology were euthanized in 0.05% Benzocaine for 30 min until no cardiac activity was observed. Specimens were fixed in 10% buffered formalin for 24 h, embedded in paraffin, sectioned, and stained with hematoxylin and eosin.

To facilitate the registration between OCT images and corresponding histology, numerous OCT images were first acquired as desired anatomical locations at 25–50 m intervals. Serial sectioning at 20 m intervals was performed during histological processing. Following light microscopic observations of the histology, OCT images from the same transverse plane in the specimen were selected in correspondence with the histological sections.

To illustrate the ability of OCT to image developing internal morphology in optically opaque specimens, a series of cross-sectional images were acquired in vitro from the dorsal and ventral sides of a stage 49 (12 day) [23] Rana pipiens tadpole. The plane of the OCT image was perpendicular to the anteroposterior axis. Figure 1 shows representative OCT images displayed in gray scale. The gray scale indicates the logarithm of the intensity of optical backscattering and spans a range of approximately 60 dB to 110 dB of the incident optical intensity. These images were 7 mm 3 mm, corresponding to 500 250 pixels with 12-bit resolution.

Figure 1 Rana pipiens tadpole. Images in left and right columns acquired from the dorsal and ventral sides, respectively. ea, ear; ey, eye; g, gills; h, heart; i, intestinal tract; m, medulla; rt, respiratory tract. Bar represents 1 mm. (From Ref. 5.)

Features of internal architectural morphology can be clearly identified in the images. Identifiable structures in Fig. 1e include the midbrain, fourth ventricle of the brain, and medulla as well as the ear vesicle. The horizontal semicircular canal and developing labyrinths are observed. Internal morphology not accessible in one orientation due to the specimen size or shadowing effects was imaged by reorienting the specimen and scanning in the same cross-sectional image plane. The images in Figs 1b, 1d, and 1f were acquired with the OCT beam incident from the ventral side to image the ventricle of the heart, internal gills, and gastrointestinal tract. The image of the eye (Fig. 1a) differentiates structures corresponding to the cornea, lens, and iris. The corneal thickness, measured to be on the order of 10 m, was resolved due to the differences in index of refraction between the water and the cornea. By imaging through the transparent lens, the incident OCT beam imaged several of the posterior ocular layers, including the ganglion cell layer, retinal neuroblasts, and choroid. The thickness of these layers were measured from the corresponding histology by using a microscope with a calibrated reticule. The thicknesses of the ganglion cell, retinal neuroblast, and choroid layers were 10 m, 80 m, and 26 m, respectively, and demonstrate the high imaging resolution of the OCT system.

Retinal layers were not imaged throughout the entire globe because of shadowing effects from the highly backscattering iris and sclera, which attenuate the transmission of light to deeper structures directly below. A sharp vertical boundary demarcated the regions where light was transmitted through the lens and where light was shadowed. Variation of the specimen orientation will vary the shadow orientation and permit the imaging of different internal structures.