optical coherence tomography imaging in developmental biology

22

Optical Coherence Tomography Imaging

in Developmental Biology

Stephen A. Boppart, Mark E. Brezinski, and James G. Fujimoto

1. Introduction

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

OCT was originally developed an9 demonstrated in ophthalmology for high-resolu-tion tomographic imaging of the retina and anterior eye (2-4). Because the eye is trans-parent 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 ofretinal pathology with 10-~ resolution, almost one order of magnitude higher thanpreviously possible using ultrasound. Clinical studies have been performed to assessthe application of OCT for a number of macular diseases (3,4). OCT is especially prom-ising for the diagnosis and monitoring of glaucoma and macular edema associated withdiabetic retinopathy because it permits the quantitative measurement of changes in theretinal or retinal-nerve fiber layer thickness. Because morphological changes oftenoccur before the onset of physical symptoms, OCT can provide a powerful approachfor the early detection of these diseases.

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

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

217

218 Boppart, Brezinski, and Fujimoto

Delayed Reflected Light/1Incident Light

Specimen

~

I

Fig. 1. OCT imaging is perfonned 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 ~or longer wavelengths. In most tissues, imaging depths of 2-3 mm can be achievedusing a system detection sensitivity of 100-110 dB. OCT has been applied to imagearterial pathology in vitro and has been shown to differentiate plaque morphology withsuperior resolution to ultrasound (10-12). Imaging studies have also been performed toinvestigate applications in gastroenterology, urology, and neurosurgery (13-15). High-resolution OCT using short-coherence-length, short-pulsed light sources has also beendemonstrated and axial resolutions of <5 ~ achieved (16,17). High-speed OCT atimage acquisition rates of 4-8 frames/s for a 250- to 500-square pixel images has beenachieved (18,19). OCT has been extended to perform Doppler imaging of blood flowand birefringence imaging to investigate laser intervention (20-22). Different imagingdelivery systems, including transverse jmaging 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 per-form in vivo imaging in animal models (25).

2. Principles of Operation

OCT is based on optical ranging, the high-resolution, high-dynamic-range detectionof backscattered light as a function of delay. In contrast to ultrasound, because thevelocity of light is extremely high, the echo time delay of reflected light cannot bemeasured directly and interferometric detection techniques must be used. One methodfor measuring echo time delay is to use low-coherence interferometry or optical-coher-ence domain reflectometry (26). Low-coherence interferometry was first developed for

Optical Coherence Tomography 219

Reference

Long Coherence Length Short Coherence Length

Fig. 2. Schematic showing the concept of low-coherence interferometry. Using a short-coherence length light source and a Michelson-type interferometer, interference fringes areobserved only when the path lengths of the two interferometer arms are matched to within acoherence length. Abbreviations: BS, beam splitter; I1lc, coherence length.

measuring reflections in fiber optics and optoelectronic devices and was demonstratedin ophthalmology for measurements of axial eye length and corneal thickness (27,28).

The echo time delay of reflected light is measured by using a Michelson-type inter-ferometer (Fig. 2). The interferometer can be implemented using a fiber optic couplerin order to yield a compact and robust system (Fig. 3). The light reflected from thespecimen or sample is interfered with light, which is reflected from a reference path ofknown path length. Interference of the light reflected from the sample arm and refer-ence arm of the interferometer can occur only when the optical path lengths of the twoarms match to within the coherence length of the optical source. As the reference-armoptical-path length is scanned, different echo delays ofbackscattered light from withinthe sample are measured. The interference signal is detected at the output port of theinterferometer, electronically bandpass filtered, demodulated, digitized, and stored ona computer. The position of the incident beam on the specimen is scanned in the trans-verse direction, and multiple axial measurements are performed. This generates a 2-Ddata array, which represents the optical backscattering through a cross-sectional planein the specimen. The logarithm of the backscatter intensity is then mapped to a falsecolor or gray scale and displayed as an OCT image (Fig. 4).

In contrast to conventional microscopy, the axial resolution in OCT images is deter-mined by the coherence length of the light source. The axial point-spread function of

220

Fig. 3. Schematic representation of OCT system implemented using fiber optics. The Michelsoninterferometer is implemented using a 3-dB (50/50) fiber coupler. The output of the interfero-meter is detected with a photodiode, demodulated, and processed by a computer.

Transverse Scanning

2D Grey Scale or False ColorImage of Optical Backscattering

Fig. 4. The OCT image is acquired by performing axial measurements of optical backscatterat different transverse positions on the specimen and displaying the resulting 2-0 data set as a

gray-scale or false-color image.


the OCT measurement as defined by the signal detected at the output of the interfero-meter is the electric-field autocorrelation of the source. The coherence length of thelight is the spatial width of the field autocorrelation, and the envelope of the fieldautocorrelation is equivalent to the Fourier transform of its power spectrum. Thus, thewidth of the autocorrelationfunction, or the axial resolution, is inversely proportionalto the width of the power spectrum. For a source with a Gaussian spectral distribution,the axial resolution Az is given by

A221n2~z=

7t L\A

where & and dA are the full-widths-at-half-maximum of the autocorrelation functionand power spectrum, respectively, and A is the source central wavelength. This meansthat high-axial resolution requires broad bandwidth optical sources.

The transverse resolution in an OCT imaging system is determined by the focusedspot size in analogy with conventional microscopy and is given by

Ax=~1t d

where d is the spot size on the objective lens andfis its focal length. High-transverseresolution can be obtained by using a large numerical aperture and focusing the beamto a small spot size. The transverse resolution is also related to the depth of focus or theconfocal parameter 2zR (two times the Raleigh range):

2ZR=~2/.'

Thus, increasing the transverse resolution produces a reduced depth of field. Typically,the confocal parameter or depth of focus is chosen to match the desired depth of imag-ing. Increased resolution may also be obtained by spatially tracking the focus.

Finally, the detection signal-to-noise ratio (SNR) is given by the optical powerbackscattered from the sample divided by the noise equivalent bandwidth (NEB):

-.!L~

M1ro NEB

SNR = 10 1og

Depending on the desired SNR performance, incident powers of 5-10 mW are typi-cally required for OCT imaging of 250-500-square pixel images at several frames persecond. If lower data-acquisition speeds or SNR can be tolerated, power requirementscan be reduced accordingly.

The majority of OCT imaging systems to date have used superluminescent diodes(SLDs) as low-coherence light sources. SLDs are commercially available at a range ofwavelengths including 800 nm, 1.3 ~, and 1.5 ~ and are attractive because they arecompact and have high efficiency and low noise. However, output powers are typicallylimited to hundreds of microwatts and the available bandwidths are relatively narrow,permitting imaging with 10-15-~ resolution. Recent advances in short-pulse solid-state laser technology make these sources attractive for OCT imaging in researchapplications. Femtosecond solid-state lasers can generate tunable, low-coherence light


at powers sufficient to permit high-speed OCT imaging. Short-pulse generation hasbeen achieved across the full wavelength range in Ti:A12O3 from 0.7-1.1 J.Un and overmore limited tuning ranges near 1.3 and 1.5 J.Un in Cr4+:Mg2SiO4 and cr4+: y AG lasers.OCT imaging with resolutions of 4 and 6 J.Un has been demonstrated at 800 nm and 1.3 J.Un,respectively, using Ti:A12O3 and Cr4+:Mg2SiO4 sources (16,17). More-compact andmore-convenient sources such as superluminescent fiber sources are currently underinvestigation (29-32). Recently, amplified SLD sources have become commerciallyavailable that have approx 15-J.Un resolutions at 1.3 J.Un and sufficient powers (> 10 m W)to permit real-time OCT imaging (AFC Technologies Inc., Hull, Quebec, Canada).

3. Methods: Developmental Biology Applications

3.1. Morphological Imaging

Imaging studies were performed on several standard biological animal models com-monly employed in developmental biology investigations (33,34). OCT imaging wasperformed in Rana pipiens tadpoles (in vitro ), Brachydanio rerio embryos and eggs(in vivo), and Xenopus laevis tadpoles (in vivo). Tadpoles were anesthetized by immer-sion in 0.05% tricaine until they no longer responded to touch. Specimens were ori-ented 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 for30 min until no cardiac activity was observed. Specimens were then fixed in 10% buff-ered formalin for 24 h, embedded in paraffin, sectioned, and stained with hematoxylinand eosin. In order to facilitate the registration between OCT images and correspond-ing histology, numerous OCT images were first acquired at desired anatomical locationsin 25-50-~ intervals between cross-sectional planes. Serial sectioning at 20-~ inter-vals was performed during histological processing. Following light microscopic obser-vations of the histology, OCT images from the same transverse plane in the specimenwere selected based on correspondence to the histological sections.

To illustrate the ability of OCT to image developing internal morphology in opti-cally opaque specimens, a series of cross-sectional images were acquired in vitro fromthe dorsal and ventral sides of a Stage 49 (12-day) R. pipiens tadpole. The plane of theOCT image was perpendicular to the anteroposterior axis. Figure 5 shows representa-tive OCT images displayed in gray scale. The gray scale indicates the logarithm of theintensity of optical backscattering and spans a range of approx -60 to -110 dB of theincident optical intensity. These images (7 x 3 mm, 500 x 250 pixels, 12-bit) were eachacquired in 40 s.

Features of internal architectural morphology are clearly visible in the images.The image of the eye (Fig. SA) differentiates structures corresponding to the cornea,lens, and iris. The corneal thickness is on the order of 10 ~ and can be resolvedbecause of the differences in index of refraction between the water and the cornea.By imaging through the transparent lens, the incident OCT beam images several of theposterior ocular layers; including the ganglion cell layer, retinal neuroblasts, and chor-oid. The thicknesses of these layers were measured from the corresponding histologyusing a microscope with a calibrated reticule. The thicknesses of the ganglion cell layer ,retinal neuroblasts, and choroid were 10,80, and 26 ~, respectively, and demonstratethe imaging resolution of the OCT system. Identifiable structures in Fig. SE include


A

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c D

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FE

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~' "~~A;';iif;c.~:,1

i';.~"c:

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Fig. 5. OCT images of Stage 49 (12-d) Rana pipiens tadpole. Images in the right column,(B), (D), and (F), were acquired with the OCT beam incident from the ventral side to imagethe respiratory tract, ventricle of the heart, internal gills, and gastrointestinal tract. Abbrevia-tions: ea, ear; ey, eye; g, gills; h, heart; i, intestine; m, medulla.

the medulla and the ear vesicle. The horizontal semicircular canal and developing laby-rinths are observed. Internal morphology not accessible in one orientation because ofthe specimen size or shadowing effects can be imaged by reorienting the specimen andscanning in the same cross-sectional image plane. The images in Fig. SB,D,F wereacquired with the OCT beam incident from the ventral side to image the respiratorytract, ventricle of the heart, internal gills, and gastrointestinal tract.

Figure 6 demonstrates the application of OCT for sequential imaging of develop-ment in vivo. Repeated images (3 x 3 mm, 300 x 300 pixels) were made of a develop-ing zebrafish embryo within its egg beginning immediately after fertilization, every 15 min,for 24 h, at the same cross-sectional plane within the specimen. Developmental changesillustrate early cleavage beginning at the two-cell stage. Because of the high mitoticrate of the embryo, results of cellular division and migration are observed as well as theestablishment of the anteroposterior axis. In this example, the zebrafish egg and embryowere semitransparent, and the use of OCT significantly complemented observationsmade using light microscopy. By imaging subtle differences in backscattering inten-sity, interfacial structural layers millimeters deep within specimens can be clearlydelineated. In Fig. 6A,B, images of the zebrafish embryo are shown at the two- andfour-cell stage, just after fertilization. Images of a zebrafish embryo prior to and imme-diately after hatching are shown in Fig. 6C,D.

~ 'i\...1" l'"c.r"c ,~~;c,:\"

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Fig. 6. Sequential OCT imaging of in vivo development in zebrafish embryo. Althoughembryo was semitransparent, the following demonstrates imaging in single specimens forextended periods oftimeo Images were acquired at (A) 1 h, (B) 1.75 h, (C) 24 h, and (D) 48 ho

3.2. Functional Imaging

Previous OCT images have characterized morphological features within biologicalspecimens. These structures are static even though they may have been acquired fromin vivo specimens. In vivo imaging in living specimens, particularly in larger organ-isms and for medical diagnostic applications, must be performed at high speeds toeliminate motion artifacts within the images. Functional imaging is the quantificationof in vivo images, which yields information characterizing the functional properties ofthe organ system or organism. High-speed OCT permits both the positioning andmanipulation of specimens as well as imaging in real time and is a powerful technologyfor functional imaging in developmental biology animal models.

Studies investigating normal and abnormal cardiac development are frequentlylimited by an inability to access cardiovascular function within the intact organism.OCT has been demonstrated for the high-resolution assessment of structure and func-tion in the developing Xenopus laevis cardiovascular system (35,36). OCT, unlike tech-


Fig. 7. (A) OCT image acquired with the diode-based system of the beating Xenopus ven-tricle. (B) OCT optical cardiogram of a normal functioning anesthetized heart. Abbreviations:EDD, end diastolic dimension; ESD, end systolic dimension; ET, ejection time; FT , filling time.

nologies such as computed tomography and magnetic resonance imaging, provideshigh-speed in vivo imaging, allowing quantitative dynamic activity, such as ventricu-lar ejection fraction, to be assessed. This is analogous to both ultrasound M-mode and

2-Dechocardiography.The super-luminescent diode-based OCT system is capable of acquiring informa-

tion of in vivo cardiovascular dynamics. Because a single axial scan can be acquired inapprox 100 ms with the diode-based system, an OCT optical cardiogram, analogous toa M-mode echocardiogram, can be obtained with this slower system. Figure 7A is anOCT image acquired with the diode-based system of the beating Xenopus ventricle.The periodic bands within the image correspond to the movement of the cardiacchamber walls during the cardiac cycle. Both the ventral and dorsal walls of the ven-tricle, in addition to the ventricular lumen, can be identified, as well as the oscillatorynature inherent during the cardiac cycle. Shown in Fig. 7A, the OCT beam was posi-tioned and held stationary over the ventricle at the site corresponding to the center ofthe image (arrow). Axial (depth) scans from this location were acquired over time andare shown in Fig. 7B, which is an OCT optical cardiogram of a normal functioninganesthetized heart.


Fig. 8. High-speed OCT images of the beating Xenopus heart. Acquisition rates of 8 frames/s

permit imaging through a single cardiac cycle.

OCT optical cardiograms permit quantitative measurements of chamber functionand allow assessment of changes over time or resulting from the administration ofpharmacological agents. Measurements included heart rate, end-diastolic and end-sys-tolic dimensions, and filling and emptying times, as illustrated in Fig. 78. From thesemeasured parameters, and by using established heart volume models, ventral wallvelocity, fractional shortening, and ejection fraction can be calculated.

Though the relatively slow data acquisition rate of the diode-based system (30 s/image )is adequate for in vitro imaging of microstructure and obtaining optical cardiogramsfrom a single location, 2-0 in vivo imaging of the rapidly beating heart requiresconsiderably faster imaging speeds. The Cr4+:forsterite laser was used as a lightsource to enable high-speedOCT imaging. Using a power of2 mW incident on thespecimen, the resulting SNR is 110 dB for high-speed imaging. Other modificationsto the OCT system include the incorporation of a new optical delay line in place ofthe mechanical galvanometer reference arm scanner. This enabled faster axial scan-ning and an image acquisition rate of 4-8 images (256 x 256 pixels) per second. Boththe axial and transverse resolutions were comparable to those for the super-Iumines-

cent diode source.In contrast to the motion artifacts present in the image in Fig. 7 A, the images in

Fig. 8 were acquired with the high-speed OCT system and are free of artifacts.The morphology of the in vivo cardiac chambers is clearly delineated at this fasterimaging speed. Image acquisition is fast enough to capture the cardiac chambers inmid-cycle. With this capability, images were acquired at various times during the car-diac cycle. A sequence of six images is shown in Fig. 8 that comprise a complete beatbeginning with the initiation of diastole, the filling of the ventricle. These frames can bedisplayed in real time to produce a movie illustrating the dynamic, functional behavior

of the developing heart.


3.3. High-Resolution Imaging

Although previous studies have demonstrated in vivo OCT imaging of tissue mor-phology, most have imaged tissue at approx 10-15 ~ resolutions, which does notallow differentiation of cellular structure. The Xenopus laevis (African frog) tadpolewas used to demonstrate the feasibility of OCT for high-resolution in vivo cellular andsubcellular imaging (37). The ability of OCT to identify the mitotic activity, the nuclear-to-cytoplasmic ratio, and the migration of cells was evaluated. OCT images were com-pared to corresponding histology to verify identified structures.

A short-pulse, solid-state, Cr4+:forsterite laser operating near 1.3 ~ was used forhigh-resolution imaging. The axial resolution was determined by the bandwidth ofthe laser source and was 5 ~. The transverse resolution was determined by the beamfocusing parameters and was 9 ~. Cells as small as 15 ~ in diameter could beimaged. Xenopus specimens ranged in age from 14 to 28 d (Stage 25-30). The speci-mens were irrigated at 5-min intervals to prevent dehydration during imaging. Mul-tiple 2-D cross sections (900 x 600 ~, 300 x 300 pixels) were acquired perpendicularto the anteroposterior axis along the entire length of the specimen. To follow cellularmitosis and migration processes, three-dimensional (3-D) OCT volumes wereacquired over time. Fifteen 2-D cross sections acquired at 5-~ intervals wereassembled to produce a 3-D dataset. Three-dimensional volumes were acquiredevery 10 min from the region posterior to the eyes and lateral to the neural tube ofthe specimen.

Immediately following image acquisition, the location of the image planes wasmarked with India ink for registration between OCT images and histology. speci-mens were euthanized by immersion in 0.05% Benzocaine for 1 h and then placed in a10% buffered solution of formaldehyde for standard histological preparation. His-tological sections, 5 ~ thick, were sectioned and stained with hematoxylin and eosinfor comparison with acquired OCT images. Correspondence was determined by thebest match between OCT images and light microscopy observations of the histol-ogy. Images were processed using IP Lab 3.0 (Signal Analytics Corp., Vienna,V A) on an Apple Computer Power Macintosh 9500/200. The 3-D volumes wereanalyzed to identify individual cells, image mitotic activity, and track migratingcells within the acquired volumes over time. Cell position was determined by mea-suring distances from two internal reference points, the neural tube and the outermembrane.

Representative high-resolution images of Xenopus architectural and cellular micro-structure are shown in Fig. 9. The arrow in Fig. 9 A identifies the intricate gill structurewithin the respiratory tract. A superficial artery less than lOO ~ in diameter is illus-trated by the arrow in Fig. 98. The lower right corner of this image reveals mesenchy-mal cells with distinct membranes and cell nuclei. A region near the nasal placode isshown in Fig. 9C. The arrows indicate pairs of daughter cells immediately followingcell division. The arrows in Fig. 9D indicate melanocytes believed to have originatedfrom the neural crest. Melanin has a high index of refraction (n = 1.7) compared withsurrounding tissue structures (n = 1.35) and therefore results in higher optical back-

scattering in OCT images. This facilitates tracking of neural crest melanocyte migra-tion deep within scattering specimens.


Fig. 9. Cellular-level OCT in vivo imaging in a developing Xenopus laevis tadpole per-formed with a high-resolution OCT system using a short-pulse laser as a low-coherence lightsource. The image resolution was 5 I.1In in the axial direction and 9 I.1In in the transverse direction.Arrows indicate (A) gill structures within respiratory tract, (B) superficial artery, (C) daughtercells immediately after cell division, and (D) melanocytes migrating from the neural crest.

4. Notes

I. The capabilities of OCT offer a unique and informative means of imaging specimens indevelopmental biology. The noncontact nature of OCT and the use of low-power, near-infrared radiation for imaging causes few harmful effects on living cells. OCT imagingdoes not require the addition of fluorophores, dyes, or stains in order to improve contrastin images. Instead, OCT relies on the inherent optical contrast generated from variationsin optical scattering and index of refraction. These factors permit the use of OCT forextended imaging of development over the course of hours, days, or weeks. As shown inFig. 6, images can be acquired over extended periods of time, in vivo, without complica-tions from toxic byproducts, which can adversely affect the viability of the developingspecimen. OCT permits the cross-sectional imaging of tissue and organ morphology andenables developing in vivo structure to be visualized in opaque specimens or in specimenstoo large for high-resolution confocal or light microscopy.

2. High-quality histology, especially serial sectioning, is often difficult, time consuming,and costly. It is especially difficult to histologically prepare the large numbers of speci-mens typically needed for genetic and developmental studies. OCT technology offers apromising alternative for rapidly accessing changes in architectural morphology and func-tional parameters. Because the position of the OCT optical beam on the specimen is pre-cisely controlled by micron-stepping-motor stages and galvanometric scanners, theregistration of the OCT imaging beam on the specimen is precisely established. Repeatedserial OCT cross-sectional images can easily be acquired to construct a 3-D representationof specimen morphology. In contrast, in histology, alignment of sectioned planes is oftendifficult and not repeatable. The major discrepancy in registration between our OCTimages and histology occurs as the result of small discrepancies in the tilt angle of theimage planes rather than axial (posterior-anterior) registration.


3. OCT functions as a type of optical biopsy that permits real-time, cross-sectional in vivoimaging of architectural morphology and functional changes. Additionally, OCT maycomplement the results obtained with histological preparations by reducing the uncer-tainty associated with artifacts often attributed to histological processing. Finally, withthe ability to detect morphological changes on the order of 5-10 J.Un, OCT offers anopportunity to identify small variations from normal development.

4. OCT image contrast results from the different optical backscattering properties betweendifferent structures. Tissue structures are differentiated according to their varying degreesof optical backscattering, whereas fluid-filled cavities within the specimen have low back-scattering. The cartilaginous skeletal system of the tadpole appears highly scattered and isclearly identified in Fig. 5A. As light propagates deeper through the specimen, a largerpercentage of the incident beam is either scattered or absorbed. Hence, less signal is avail-able from deeper structures, and in a gray-scale image, the image becomes lighter as theSNR is reduced. Morphology located directly below a highly backscattering structure canbe shadowed by the structure above it. For example, in Fig. 5, retinal layers are not imagedthroughout the entire globe because of shadowing effects from the highly backscatteringiris and sclera, which attenuate the transmission of light to deeper structures directly below.A sharp vertical boundary demarcates the regions where light is transmitted through thelens and where light is shadowed. Variation of the specimen orientation will vary theshadow orientation and permit the imaging of different internal structures. These effectsare analogous to attenuation and shadowing observed in ultrasound. If the biological speci-men is relatively homogeneous, the signal attenuation with depth can be compensated bysimple image-processing techniques. However, because the morphology of the specimensused in this study is complex, these techniques were not applied here.

5. A number of comparisons and contrasts can be drawn between the OCT images and histo-logical preparations. There is a strong correlation of the tissue architectural morphologybetween OCT images and the histology. However, it is important to note that OCT imagestissue properties in a completely different manner than does histology. Histology relies onthe differences in the transmission of light through stained tissue while OCT relies ondifferentials in optical backscattering. Histology with light and electron microscopy offersunprecedented resolution on the cellular and subcellular level. OCT does not have compa-rable resolution but has the ability to rapidly and repeatedly perform imaging in vivo.Histological images have artifacts caused by tissue dehydration, shrinkage, and stretchingduring processing. OCT images have artifacts that arise from optical attenuation, withdepth, shadowing, and refractive index effects. The axial distances measured in OCTimages represent the echo time delay of light, and thus, in order to convert this informa-tion into physical dimensions, it is necessary to know the index of refraction of the tissue.The index of most tissues varies between 1.35 and 1.45; thus, possible errors in longitudi-nal range can be of the order of only 5-10% if the index is unknown.

6. In addition to axial scale changes, the index of refraction also produces refraction of lightrays when they traverse boundaries with different indexes. This effect is most significantat the proximal boundary of the specimen where the OCT beam is incident on the speci-men from air. This refraction effect can cause internal features to appear as if they areangularly displaced. Refraction depends on the mismatch of the index across a boundaryand is negligible if the light enters the tissue perpendicular to the boundary and becomeslarger when the light ray is more oblique. These errors can be minimized by either par-tially or fully submerging the specimens in liquid, which produces refractive index match-ing. It is important to note that these same scale uncertainties and refractive effects arealso present in ultrasound imaging. However, if measurements are performed in a consis-tent manner, these effects are considered part of the baseline. The diagnostic power of


the imaging technique is not compromised, because it relies on detecting deviationsfrom the baseline.

7. The image scale and distortion effects are actually present in both ultrasound and MRIimages, although they arise from slightly different physics principles. Histology, to someextent, suffers from similar problems in obtaining measurements from preparationsresulting from tissue preservation, dehydration, and sectioning. Tissue configuration fol-lowing preparation may not always reflect the in vivo orientation, making quantificationdifficult. Despite the artifacts in both techniques, these artifacts are reproducible and canbe perceived as the baseline. These differences in calibrating histopathology against real-tissue dimensions usually do not compromise diagnostic utility.

8. Imaging at cellular and subcellular resolutions with OCT is an important area of ongoingresearch. The Xenopus developmental animal model was selected because its care andhandling are relatively simple, at the same time allowing cells with a high mitotic index tobe assessed. Many of the cells observed were as large as 100 I.IIn in diameter, but ranged insize down to a few microns, below the resolution of this OCT system. Precise imageregistration with histology is more problematic at the cellular level. In addition to theproblem of registering the OCT image with histology, cells may divide, grow, andmove between the time the cells are imaged and the time the specimen is euthanized andfixed for processing. However, high-speed imaging at cellular resolutions should permitreal-time tracking of a cell. The ability to position the OCT imaging plane at arbitraryangles is advantageous, especially when cell division is to be observed. The division of acell into two daughter cells may not always occur in a predicted plane.

9. With further advances in OCT technology, improved discrimination and imaging of more-detailed morphology should be possible. New laser sources at other wavelengths in thenear-infrared can enhance tissue contrast as well as potentially provide functional infor-mation, because tissue scattering and absorbance properties in specimens are wavelengthdependent. Short-coherence-length short-pulse laser sources have been used to achievehigher axial resolutions on the order of 2-4 1.IIn. Unfortunately, unlike superluminescentdiode sources, these high-speed and high-resolution systems utilize femtoscond lasers,which are relatively complex and costly. As noted previously, simpler and more economi-cal sources such as superluminescent fibers are currently under development. OCT can beperformed using high numerical aperture lenses to achieve high transverse resolutions, asin confocal microscopy, at the expense of reducing the depth of field. Real-time imageacquisition has been demonstrated. High-speed imaging reduces specimen motion arti-facts and should allow images to be obtained without the need for anesthesia. Imagingacquisition speeds may be further increased by increasing source power and scanningspeed. In combination with specially designed catheters or endoscopes, in utero imaging ofembryonic and fetal development in live-bearing species may be possible.

10. Imaging developing embryonic morphology with OCT offers many new possibilities forstudies in developmental biology as well as for microscopy in general. Optical coherencetomography provides high-resolution morphological, functional, and cellular informationon developing biology in vivo. These results demonstrate the feasibility of gaining furtherinsight into the morphological and functional expression of the genetic program, High-resolution, in vivo optical imaging using OCT permits the identification of morphologicalvariations during embryogenesis. By tracking the results of cellular differentiation andrapidly identifying normal and abnormal organo- and morphogenesis in embryos, OCTrepresents a multifunctional investigative tool that should not only complement many ofthe existing imaging technologies available today, but will also reveal previously unseen

dynamic changes during development.


AcknowledgmentsWe would like to thank Dr. Hazel Sive and her associates at the Massachusetts Insti-

tute of Technology for invaluable scientific advice and assistance. We also acknowl-edge the contributions of Drs. Brett Bouma and Gary Teamey. This research wassupported in part by NIH contracts ROI-EYl1289, ROI-CA75289, and the Office ofNaval Research contract NO 0014-97-1-1066.

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optical coherence tomography imaging in developmental biology

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