Optical coherence tomography.

Optical coherence tomography (OCT) is a non-contact method for imaging the topological and internal microstructure of samples in three dimensions. OCT can be configured as a conventional microscope, as an ophthalmic scanner, or using endoscopes and small diameter catheters for accessing internal biological organs. In this Primer, we describe the principles underpinning the different instrument configurations that are tailored to distinct imaging applications and explain the origin of signal, based on light scattering and propagation. Although OCT has been used for imaging inanimate objects, we focus our discussion on biological and medical imaging. We examine the signal processing methods and algorithms that make OCT exquisitely sensitive to reflections as weak as just a few photons and that reveal functional information in addition to structure. Image processing, display and interpretation, which are all critical for effective biomedical imaging, are discussed in the context of specific applications. Finally, we consider image artifacts and limitations that commonly arise and reflect on future advances and opportunities.

Correlation of localized glaucomatous visual field defects and spectral domain optical coherence tomography retinal nerve fiber layer thinning using a modified structure-function map for OCT.

PURPOSE: To study the correlation between glaucomatous visual field (VF) defects assessed by standard automated perimetry (SAP) and peripapillary retinal nerve fiber layer (RNFL) thinning measured by spectral domain optical coherence tomography (OCT) using a modified OCT-based peripapillary RNFL structure-function map. PATIENTS AND METHODS: Perimetric glaucoma patients and age-matched normal control subjects were recruited from a university hospital clinic. All eyes underwent testing with the Spectralis spectral domain OCT and SAP on the same day. An OCT-based correspondence map, which correlated VF areas with peripapillary RNFL sectors was created to evaluate the relationship between glaucomatous RNFL thinning and VF loss in six nerve fiber layer bundle areas. Correlations of RNFL thinning with corresponding VF defects were examined using Spearman rank-order correlations. To demonstrate the association between localized VF defects and RNFL thickness, the theoretical curves were made according to an established log-linear model. The measured RNFL thickness values and VF defects were presented in the same scatterplot for each sector. RESULTS: Fifty-six glaucoma patients and 85 normal subjects were included in the study. Significant association between localized VF loss and RNFL thinning was found in corresponding areas. Data from the current study fit well with established log-linear models, which compare RNFL thickness values with VF defects. CONCLUSION: Analysis of RNFL thinning in eyes with localized glaucomatous VF defects showed good structure-function correlation in a new OCT-based structure-function correspondence map.

Depth-resolved model-based reconstruction of attenuation coefficients in optical coherence tomography.

We present a method, based on a single scattering model, to calculate the attenuation coefficient of each pixel in optical coherence tomography (OCT) depth profiles. Numerical simulations were used to determine the model's response to different depths and attenuation coefficients. Experiments were performed on uniform and layered phantoms with varying attenuation coefficients. They were measured by a 1300 nm OCT system and their attenuation coefficients were evaluated by our proposed method and by fitting the OCT slope as the gold standard. Both methods showed largely consistent results for the uniform phantoms. On the layered phantom, only our proposed method accurately estimated the attenuation coefficients. For all phantoms, the proposed method largely reduced the variability of the estimated attenuation coefficients. The method was illustrated on an in-vivo retinal OCT scan, effectively removing common imaging artifacts such as shadowing. By providing localized, per-pixel attenuation coefficients, this method enables tissue characterization based on attenuation coefficient estimates from OCT data.

Automated segmentation by pixel classification of retinal layers in ophthalmic OCT images.

Current OCT devices provide three-dimensional (3D) in-vivo images of the human retina. The resulting very large data sets are difficult to manually assess. Automated segmentation is required to automatically process the data and produce images that are clinically useful and easy to interpret. In this paper, we present a method to segment the retinal layers in these images. Instead of using complex heuristics to define each layer, simple features are defined and machine learning classifiers are trained based on manually labeled examples. When applied to new data, these classifiers produce labels for every pixel. After regularization of the 3D labeled volume to produce a surface, this results in consistent, three-dimensionally segmented layers that match known retinal morphology. Six labels were defined, corresponding to the following layers: Vitreous, retinal nerve fiber layer (RNFL), ganglion cell layer & inner plexiform layer, inner nuclear layer & outer plexiform layer, photoreceptors & retinal pigment epithelium and choroid. For both normal and glaucomatous eyes that were imaged with a Spectralis (Heidelberg Engineering) OCT system, the five resulting interfaces were compared between automatic and manual segmentation. RMS errors for the top and bottom of the retina were between 4 and 6 µm, while the errors for intra-retinal interfaces were between 6 and 15 µm. The resulting total retinal thickness maps corresponded with known retinal morphology. RNFL thickness maps were compared to GDx (Carl Zeiss Meditec) thickness maps. Both maps were mostly consistent but local defects were better visualized in OCT-derived thickness maps.

Spectral domain optical coherence tomography for quantitative evaluation of drusen and associated structural changes in non-neovascular age-related macular degeneration.

BACKGROUND/AIMS: To demonstrate how spectral domain optical coherence tomography (SDOCT) can better evaluate drusen and associated anatomical changes in eyes with non-neovascular age-related macular degeneration (AMD) compared with time domain optical coherence tomography (TDOCT). METHODS: Images were obtained from three eyes of three patients with AMD using an experimental SDOCT system. Both a titanium-sapphire (Ti:sapphire) laser and a superluminescent diode (SLD) were used as a broadband light source to achieve cross-sectional images of the retina. A qualitative and quantitative analysis was performed for structural changes associated with non-neovascular AMD. An automated algorithm was developed to analyse drusen area and volume from SDOCT images. TDOCT was performed using the fast macular scan (StratusOCT, Carl Zeiss Meditec, Dublin, California). RESULTS: SDOCT images can demonstrate structural changes associated with non-neovascular AMD. A new SDOCT algorithm can determine drusen area, drusen volume and proportion of drusen. CONCLUSIONS: With new algorithms to determine drusen area and volume and its unprecedented simultaneous ultra-high speed ultra-high resolution imaging, SDOCT can improve the evaluation of structural abnormalities in non-neovascular AMD.

Application of maximum likelihood estimator in nano-scale optical path length measurement using spectral-domain optical coherence phase microscopy.

Spectral-domain optical coherence phase microscopy (SD-OCPM) measures minute phase changes in transparent biological specimens using a common path interferometer and a spectrometer based optical coherence tomography system. The Fourier transform of the acquired interference spectrum in spectral-domain optical coherence tomography (SD-OCT) is complex and the phase is affected by contributions from inherent random noise. To reduce this phase noise, knowledge of the probability density function (PDF) of data becomes essential. In the present work, the intensity and phase PDFs of the complex interference signal are theoretically derived and the optical path length (OPL) PDF is experimentally validated. The full knowledge of the PDFs is exploited for optimal estimation (Maximum Likelihood estimation) of the intensity, phase, and signal-to-noise ratio (SNR) in SD-OCPM. Maximum likelihood (ML) estimates of the intensity, SNR, and OPL images are presented for two different scan modes using Bovine Pulmonary Artery Endothelial (BPAE) cells. To investigate the phase accuracy of SD-OCPM, we experimentally calculate and compare the cumulative distribution functions (CDFs) of the OPL standard deviation and the square root of the Cramér-Rao lower bound (1/ square root 2SNR ) over 100 BPAE images for two different scan modes. The correction to the OPL measurement by applying ML estimation to SD-OCPM for BPAE cells is demonstrated.

High-speed polarization sensitive optical frequency domain imaging with frequency multiplexing.

Polarization sensitive optical coherence tomography (PS-OCT) provides a cross-sectional image of birefringence in biological samples that is complementary in many applications to the standard reflectance-based image. Recent ex vivo studies have demonstrated that birefringence mapping enables the characterization of collagen and smooth muscle concentration and distribution in vascular tissues. Instruments capable of applying these measurements percutaneously in vivo may provide new insights into coronary atherosclerosis and acute myocardial infarction. We have developed a polarization sensitive optical frequency domain imaging (PS-OFDI) system that enables high-speed intravascular birefringence imaging through a fiber-optic catheter. The novel design of this system utilizes frequency multiplexing to simultaneously measure reflectance of two incident polarization states, overcoming concerns regarding temporal variations of the catheter fiber birefringence and spatial variations in the birefringence of the sample. We demonstrate circular cross-sectional birefringence imaging of a human coronary artery ex vivo through a flexible fiber-optic catheter with an A-line rate of 62 kHz and a ranging depth of 6.2 mm.

Large depth-high resolution full 3D imaging of the anterior segments of the eye using high speed optical frequency domain imaging.

Three dimensional rapid large depth range imaging of the anterior segments of the human eye by an optical frequency domain imaging system is presented. The tunable source spans from 1217 to 1356 nm with an average output power of 60 mW providing a measured axial resolution of 10 mum in air based on the coherence envelope. The effective depth range is 4 mm, defined as the distance over which the sensitivity drops by 6 dB, achieved by frequency shifting the optical signal using acousto-optic modulators. The measured maximum sensitivity is 109 dB at a sample arm power of 14.7mW and A-lines rate of 43,900 per second. Images consisting of 512 depth profiles are acquired at an acquisition rate of 85 frames per second. We demonstrate an optical frequency domain imaging system capable of mapping in vivo the entire area of the human anterior segment (13.4 x 12 x 4.2 mm) in 1.4 seconds.

In vivo thickness and birefringence determination of the human retinal nerve fiber layer using polarization-sensitive optical coherence tomography.

Thinning of the retinal nerve fiber layer and changes in retinal nerve fiber layer birefringence may both precede clinically detectable glaucomatous vision loss. We present in vivo thickness and depth-resolved birefringence measurements of the human retinal nerve fiber layer (RNFL) by use of polarization-sensitive optical coherence tomography (PS-OCT). Using a fiber-based PS-OCT setup real-time images of the human retina in vivo were recorded, co-registered with retinal video images of the location of PS-OCT scans. PS-OCT scans around the optic nerve head (ONH) of two healthy young volunteers were made using 10 concentric circles of increasing radius. Both the mean retinal nerve fiber layer thickness and mean retinal nerve fiber birefringence for each of 48 sectors on a circle were determined. The retinal nerve fiber layer thickness and birefringence varied as a function of sector around the ONH. Measured double pass phase retardation per unit depth values around the ONH range between 0.10 and 0.35 degrees/microm. The retinal nerve fiber layer becomes thinner with increasing distance from the ONH. In contrast, the birefringence does not vary significantly with increasing distance from the ONH.

Ultra-high speed and ultra-high resolution spectral-domain optical coherence tomography and optical Doppler tomography in ophthalmology.

We present ultra-high resolution optical coherence tomography (OCT) structural intensity and optical Doppler tomography (ODT) flow velocity images of the human retina in vivo. The ultra-high speed OCT system is based on Spectral Domain or Fourier Domain technology, which provides a sensitivity advantage over conventional OCT of more than 2 orders of magnitude. This sensitivity improvement allows video rate OCT and ODT cross sectional imaging of retinal structures. Images will be presented with axial resolutions of 6 and 3.5 microns. We observed small features in the inner and outer plexiform layers, which are believed to be small blood vessels. Flow velocity images will be presented showing pulsatile flow in retinal arteries and veins.

Optical frequency domain imaging with a rapidly swept laser in the 815-870 nm range.

Optical frequency domain imaging (OFDI) in the 800-nm biological imaging window is demonstrated by using a novel wavelength-swept laser source. The laser output is tuned continuously from 815 to 870 nm at a 43.2-kHz repetition rate with 7-mW average power. Axial resolution of 10-mum in biological tissue and peak sensitivity of 96 dB are achieved. In vivo imaging of Xenopus laevis is demonstrated with an acquisition speed of 84 frames per second (512 axial lines per frame). This new imaging technique may prove useful in comprehensive retinal screening for medical diagnosis and contrast-agent-based imaging for biological investigations.

High-speed imaging of human retina in vivo with swept-source optical coherence tomography.

We present the first demonstration of human retinal imaging in vivo using optical frequency domain imaging (OFDI) in the 800-nm range. With 460-muW incident power on the eye, the sensitivity is 91 dB at maximum and >85 dB over 2-mm depth range. The axial resolution is 13 mum in air. We acquired images of retina at 43,200 depth profiles per second and a continuous acquisition speed of 84 frames/s (512 A-lines per frame) could be maintained over more than 2 seconds.

Extended-Cavity Semiconductor Wavelength-Swept Laser for Biomedical Imaging.

We demonstrate a compact high-power rapidly swept wavelength tunable laser source based on a semiconductor optical amplifier and an extended-cavity grating filter. The laser produces excellent output characteristics for biomedical imaging, exhibiting >4-mW average output power, <0.06-nm instantaneous linewidth, and >80-dB noise extinction with its center wavelength swept over 100 nm at 1310 nm at variable repetition rates up to 500 Hz.

Visualizing early frog development with motion-sensitive 3-D optical coherence microscopy.

A motion-sensitive en-face-scanning 3-D optical coherence microscope (OCM) has been designed and constructed to study critical events in the early development of plants and animals. We describe the OCM instrument and present time-lapse movies of frog gastrulation, an early developmental event in which three distinct tissue layers are established that later give rise to all major organ systems. OCM images constructed with fringe-amplitude data show the mesendoderm migrating up along the blastocoel roof, thus forming the inner two tissue layers. Motion-sigma data, measuring the random motion of scatterers, is used to construct complementary images that indicate the presence of Brownian motion in the yolk cells of the endoderm. This random motion provides additional intrinsic contrast that helps to distinguish different tissue types. Depth penetration at 850 nm is sufficient for studies of the outer ectoderm layer, but is not quite adequate for detailed study of the blastocoel floor, about 500 to 800 mum deep into the embryo. However, we measure the optical attenuation of these embryos to be about 35% less at 1310 nm. 2-D OCT images at 1310 nm are presented that promise sufficient depth penetration to test current models of cell movement near the blastocoel floor during gastrulation.

In vivo burn depth determination by high-speed fiber-based polarization sensitive optical coherence tomography.

We report the first application of high-speed fiber-based polarization sensitive optical coherence tomography (PS-OCT) to image burned tissue in vivo. Thermal injury denatures collagen in skin and PS-OCT can measure the reduction in collagen birefringence using depth resolved changes in the polarization state of light propagated in, and reflected from, the tissue. Stokes vectors were calculated for each point in a scan and birefringence relative to incident polarization determined using four incident polarization states. Using a high-speed fiber-based PS-OCT system on rat skin burned for varying periods of time, a correlation between birefringence and actual burn depth determined by histological analysis was established. In conclusion, PS-OCT has potential use for noninvasive assessment of burn depth.

Stable carrier generation and phase-resolved digital data processing in optical coherence tomography.

We present a technique for improved carrier generation by eliminating the instability of a mechanical device in favor of an electro-optical phase modulator in the reference arm of an optical coherence tomography system. A greater than threefold reduction in the phase variance between consecutive A-line scans at a repetition rate of 1 kHz was achieved. Stable and reproducible interference fringe generation permits phase-resolved digital data processing. A correction algorithm was applied to the interferometric signal to compensate for the departure of the source spectrum from an ideal Gaussian shape, resulting in up to 8-dB sidelobe suppression at the expense of a 1-dB increase in the noise floor. In addition, we could eliminate completely the broadening effect of group-delay dispersion on the coherence function by introducing a quadratic phase shift in the Fourier domain of the interferometric signal.

Optical coherence tomography of the rat cochlea.

Optical coherence tomography (OCT) was used to image the internal structure of a rat cochlea (ex vivo). Immediately following sacrifice, the temporal bone of a Sprague-Dawley rat was harvested. Axial OCT cross sectional images (over regions of interest, 1x1 mm-2x8 mm) were obtained with a spatial resolution of 10-15 microm. The osseous borders of the lateral membranous labyrinth overlying the cochlea and the scala vestibuli, media, and tympani, which were well demarcated by the modiolus, Reissner's and the basilar membranes, were clearly identified. OCT can be used to image internal structures in the cochlea without violating the osseous labyrinth using simple surgical exposure of the promontory, and may potentially be used to diagnose inner ear pathology in vivo in both animal and human subjects labyrinth.

Phase-resolved optical coherence tomography and optical Doppler tomography for imaging blood flow in human skin with fast scanning speed and high velocity sensitivity.

We have developed a novel phase-resolved optical coherence tomography (OCT) and optical Doppler tomography (ODT) system that uses phase information derived from a Hilbert transformation to image blood flow in human skin with fast scanning speed and high velocity sensitivity. Using the phase change between sequential scans to construct flow-velocity imaging, this technique decouples spatial resolution and velocity sensitivity in flow images and increases imaging speed by more than 2 orders of magnitude without compromising spatial resolution or velocity sensitivity. The minimum flow velocity that can be detected with an axial-line scanning speed of 400 Hz and an average phase change over eight sequential scans is as low as 10 microm/s, while a spatial resolution of 10 microm is maintained. Using this technique, we present what are to our knowledge the first phase-resolved OCT/ODT images of blood flow in human skin.

High-speed fiber based polarization-sensitive optical coherence tomography of in vivo human skin.

A high-speed single-mode fiber-based polarization-sensitive optical coherence tomography (PS OCT) system was developed. With a polarization modulator, Stokes parameters of reflected flight for four input polarization states are measured as a function of depth. A phase modulator in the reference arm of a Michelson interferometer permits independent control of the axial scan rate and carrier frequency. In vivo PS OCT images of human skin are presented, showing subsurface structures that are not discernible in conventional OCT images. A phase retardation image in tissue is calculated based on the reflected Stokes parameters of the four input polarization states.

Doppler standard deviation imaging for clinical monitoring of in vivo human skin blood flow.

We used a novel phase-resolved optical Doppler tomographic (ODT) technique with very high flow-velocity sensitivity (10microm/s) and high spatial resolution (10microm) to image blood flow in port-wine stain (PWS) birthmarks in human skin. In addition to the regular ODT velocity and structural images, we use the variance of blood flow velocity to map the PWS vessels. Our device combines ODT and therapeutic systems such that PWS blood flow can be monitored in situ before and after laser treatment. To the authors' knowledge this is the first clinical application of ODT to provide a fast semiquantitative evaluation of the efficacy of PWS laser therapy in situ and in real time.