Differentiation of morphotic elements in human blood using optical coherence tomography and a microfluidic setup.

We demonstrate a novel optical method for the detection and differentiation between erythrocytes and leukocytes that uses amplitude and phase information provided by optical coherence tomography (OCT). Biological cells can introduce significant phase modulation with substantial scattering anisotropy and dominant forward-scattered light. Such physical properties may favor the use of a trans-illumination imaging technique. However, an epi-illumination mode may be more practical and robust in many applications. This study describes a new way of measuring the phase modulation introduced by flowing microobjects. The novel part of this invention is that it uses the backscattered signal from the substrate located below the flowing/moving objects. The identification of cells is based on phase-sensitive OCT signals. To differentiate single cells, a custom-designed microfluidic device with a highly scattering substrate is introduced. The microchannels are molded in polydimethylsiloxane (PDMS) mixed with titanium dioxide (TiO2) to ensure high scattering properties. The statistical parameters of the measured signal depend on the cells' features, such as their size, shape, and internal structure.

Red blood cell distribution width is associated with increased interactions of blood cells with vascular wall.

The mechanism underlying the association between elevated red cell distribution width (RDW) and poor prognosis in variety of diseases is unknown although many researchers consider RDW a marker of inflammation. We hypothesized that RDW directly affects intravascular hemodynamics, interactions between circulating cells and vessel wall, inducing local changes predisposing to atherothrombosis. We applied different human and animal models to verify our hypothesis. Carotid plaques harvested from patients with high RDW had increased expression of genes and proteins associated with accelerated atherosclerosis as compared to subjects with low RDW. In microfluidic channels samples of blood from high RDW subjects showed flow pattern facilitating direct interaction with vessel wall. Flow pattern was also dependent on RDW value in mouse carotid arteries analyzed with Magnetic Resonance Imaging. In different mouse models of elevated RDW accelerated development of atherosclerotic lesions in aortas was observed. Therefore, comprehensive biological, fluid physics and optics studies showed that variation of red blood cells size measured by RDW results in increased interactions between vascular wall and circulating morphotic elements which contribute to vascular pathology.

High-resolution, ultrafast, wide-field retinal eye-tracking for enhanced quantification of fixational and saccadic motion.

We introduce a novel, noninvasive retinal eye-tracking system capable of detecting eye displacements with an angular resolution of 0.039 arcmin and a maximum velocity of 300°/s across an 8° span. Our system is designed based on a confocal retinal imaging module similar to a scanning laser ophthalmoscope. It utilizes a 2D MEMS scanner ensuring high image frame acquisition frequencies up to 1.24 kHz. In contrast with leading eye-tracking technology, we measure the eye displacements via the collection of the observed spatial excursions for all the times corresponding a full acquisition cycle, thus obviating the need for both a baseline reference frame and absolute spatial calibration. Using this approach, we demonstrate the precise measurement of eye movements with magnitudes exceeding the spatial extent of a single frame, which is not possible using existing image-based retinal trackers. We describe our retinal tracker, tracking algorithms and assess the performance of our system by using programmed artificial eye movements. We also demonstrate the clinical capabilities of our system with in vivo subjects by detecting microsaccades with angular extents as small as 0.028°. The rich kinematic ocular data provided by our system with its exquisite degree of accuracy and extended dynamic range opens new and exciting avenues in retinal imaging and clinical neuroscience. Several subtle features of ocular motion such as saccadic dysfunction, fixation instability and abnormal smooth pursuit can be readily extracted and inferred from the measured retinal trajectories thus offering a promising tool for identifying biomarkers of neurodegenerative diseases associated with these ocular symptoms.

Three-dimensional quantitative imaging of retinal and choroidal blood flow velocity using joint Spectral and Time domain Optical Coherence Tomography.

Recently, joint Spectral and Time domain Optical Coherence Tomography (joint STdOCT) has been proposed to measure ocular blood flow velocity. Limitations of CCD technology allowed only for two-dimensional imaging at that time. In this paper we demonstrate fast three-dimensional STdOCT based on ultrahigh speed CMOS camera. Proposed method is straightforward, fully automatic and does not require any advanced image processing techniques. Three-dimensional distributions of axial velocity components of the blood in human eye vasculature are presented: in retinal and, for the first time, in choroidal layer. Different factors that affect quality of velocity images are discussed. Additionally, the quantitative measurement allows to observe a new interesting optical phenomenon - random Doppler shift in OCT signals that forms a vascular pattern at the depth of sclera.

Flow velocity estimation by complex ambiguity free joint Spectral and Time domain Optical Coherence Tomography.

We show that recently introduced joint Spectral and Time domain Optical Coherence Tomography (STdOCT) can be used for simultaneous complex ambiguity removal and functional Spectral OCT images. This permits to take advantage of higher sensitivity achievable near the zero-path delay. The technique can be used with all Spectral OCT systems that are equipped with an optical delay line (ODL) and provide oversampled scanning patterns. High sensitivity provided by STdOCT allows this technique to be used in Spectral OCT setups with acquisition speed of 100,000 lines/s. We show that different imaging ranges and velocity ranges can be achieved by switching on/off the ODL and a small modification in the processing algorithm. Additionally, the relatively small computational burden of the technique allows for fast computations in the range of less than 5 minutes for 3D data set. We present application of proposed technique to full-range two- and three-dimensional imaging. Morphological and Doppler tomograms of human retina in-vivo are shown. Finally, we identify and discuss artifacts of the technique.

Scanning protocols dedicated to smart velocity ranging in spectral OCT.

We introduce a new type of scanning protocols, called segmented protocols, which enable extracting multi-range flow velocity information from a single Spectral OCT data set. The protocols are evaluated using a well defined flow in a glass capillary. As an example of in vivo studies, we demonstrate two- and three-dimensional imaging of the retinal vascular system in the eyes of healthy volunteers. The flow velocity detection is performed using a method of Joint Spectral and Time domain OCT. Velocity ranging is demonstrated in imaging of retinal vasculature in the macular region and in the optic disk area characterized by different flow velocity values. Additionally, an enhanced visualization of retinal capillary network is presented in the close proximity to macula.

Fuchs' endothelial dystrophy in 830-nm spectral domain optical coherence tomography.

This article presents for the first time the potential of 830-nm spectral domain optical coherence tomography (SD-OCT) in the evaluation of Fuchs' endothelial dystrophy. SD-OCT is an imaging technique that can be used for in vivo cross-sectional corneal visualization. The important features of SD-OCT instruments include improved sensitivity and short acquisition time, which improves the quality of the tomograms compared with conventional time domain OCT. Tomograms of the corneas of three patients in different stages of Fuchs' endothelial dystrophy are presented. The authors conclude that 830-nm SD-OCT provides clinically valuable cross-sectional assessment of pathomorphological changes in Fuchs' endothelial dystrophy in vivo.

Analysis of the outer retina reconstructed by high-resolution, three-dimensional spectral domain optical coherence tomography.

BACKGROUND AND OBJECTIVE: A retrospective cross-sectional study was conducted to demonstrate an analysis of an outer retinal layer reconstructed by the three-dimensional and high-speed spectral domain optical coherence tomography (SD-OCT) instrument. PATIENTS AND METHODS: New measurement protocols for SD-OCT and methods of analysis and visualization of the individual segmented retinal layer reconstructed by SD-OCT were proposed. Three contour maps representing mutual distances between the basal part of the retinal pigment epithelium, the junction between the inner and outer segments of photoreceptors, and a reference contour representing the shape of a healthy retina were introduced. RESULTS: The analysis of the outer retina was performed on pathological eyes. Three cases of central serous chorioretinopathy, age-related macular degeneration, and acute zonal occult outer retinopathy are demonstrated. CONCLUSION: Three contour maps reconstructed for clinical cases demonstrate high variability of observed patterns depending on analyzed pathology. The authors believe this can help to present OCT data simultaneously in a more comprehensive and convenient way to assist in everyday clinical diagnosis.

Quality improvement of OCT angiograms with elliptical directional filtering.

We present a method of OCT angiography (OCTA) data filtering for noise suppression and improved visualization of the retinal vascular networks in en face projection images. In our approach, we use a set of filters applied in three orthogonal axes in the three-dimensional (3-D) data sets. Minimization of artifacts generated in B-scan-wise data processing is accomplished by filtering the cross-sections along the slow scanning axis. A-scans are de-noised by axial filtering. The core of the method is the application of directional filtering to the C-scans, i.e. one-pixel thick sections of the 3-D data set, perpendicular to the direction of the scanning OCT beam. The method uses a concept of structuring, directional kernels of shapes matching the geometry of the image features. We use rotating ellipses to find the most likely local orientation of the vessels and use the best matching ellipses for median filtering of the C-scans. We demonstrate our approach in the imaging of a normal human eye with laboratory-grade spectral-domain OCT setup. The "field performance" is demonstrated in imaging of diabetic retinopathy cases with a commercial OCT device. The absolute complex differences method is used for the generation of OCTA images from the data collected in the most noise-wise unfavorable OCTA scanning regime-two frame scanning.

Flow velocity estimation using joint Spectral and Time domain Optical Coherence Tomography.

We propose a modified method of acquisition and analysis of Spectral Optical Coherence Tomography (SOCT) data to provide information about flow velocities. The idea behind this method is to acquire a set of SOCT spectral fringes dependent on time followed by a numerical analysis using two independent Fourier transformations performed in time and optical frequency domains. Therefore, we propose calling this method as joint Spectral and Time domain Optical Coherence Tomography (joint STdOCT). The flow velocities obtained by joint STdOCT are compared with the ones obtained by known, phase-resolved SOCT. We observe that STdOCT estimation is more robust for measurements with low signal to noise ratio (SNR) as well as in conditions of close-to-limit velocity measurements. We also demonstrate that velocity measurement performed with STdOCT method is more sensitive than the one obtained by the phase-resolved SOCT. The method is applied to biomedical imaging, in particular to in vivo measurements of retinal blood circulation. The applicability of STdOCT different measurement modes for in vivo examinations, including 1, 5 and 40 mus of CCD exposure time, is discussed.

Improved spectral optical coherence tomography using optical frequency comb.

We identify and analyze factors influencing sensitivity drop-off in Spectral OCT and propose a system employing an Optical Frequency Comb (OFC) to verify this analysis. Spectral Optical Coherence Tomography using a method based on an optical frequency comb is demonstrated. Since the spectrum sampling function is determined by the comb rather than detector pixel distribution, this method allows to overcome limitations of high resolution Fourier-domain OCT techniques. Additionally, the presented technique also enables increased imaging range while preserving high axial resolution. High resolution cross-sectional images of biological samples obtained with the proposed technique are presented.

Analysis of posterior retinal layers in spectral optical coherence tomography images of the normal retina and retinal pathologies.

We present a computationally efficient, semiautomated method for analysis of posterior retinal layers in three-dimensional (3-D) images obtained by spectral optical coherence tomography (SOCT). The method consists of two steps: segmentation of posterior retinal layers and analysis of their thickness and distance from an outer retinal contour (ORC), which is introduced to approximate the normal position of external interface of the healthy retinal pigment epithelium (RPE). The algorithm is shown to effectively segment posterior retina by classifying every pixel in the SOCT tomogram using the similarity of its surroundings to a reference set of model pixels from user-selected area(s). Operator intervention is required to assess the quality of segmentation. Thickness and distance maps from the segmented layers and their analysis are presented for healthy and pathological retinas.

In vivo imaging of posterior capsule opacification using Spectral Optical Coherence Tomography.

Three years after uneventful extracapsular cataract extraction with implantation of a poly(methyl methacrylate) (PMMA) intraocular lens (IOL) (MZ60BD, Alcon) in a 74-year-old man, the anterior segment of the right eye was photographed with a Canon EOS 300D digital camera and examined with a slitlamp and a prototype spectral optical coherence tomography (SOCT) instrument. Subsequently, a neodymium:YAG laser posterior capsulotomy was performed, followed by another examination. The quality of the SOCT images was adequate for detailed cross-sectional evaluation of the IOL, posterior capsule opacification (PCO), and morphological changes after laser capsulotomy. En face contour maps of PCO distribution were created from 3-dimensional SOCT data. The results presented indicate future applicability of SOCT technology in evaluating modern IOL designs and investigating the process of PCO formation.

Spectral optical coherence tomography: a novel technique for cornea imaging.

PURPOSE: Spectral optical coherence tomography (SOCT) is a new, noninvasive, noncontact, high-resolution technique that provides cross-sectional images of the objects that weakly absorb and scatter light. SOCT, because of very short acquisition time and high sensitivity, is capable of providing tomograms of substantially better quality than the conventional OCT. The aim of this paper is to show the application of the SOCT to cross-sectional imaging of the cornea and its pathologies. METHODS: Eleven eyes with different corneal pathologies were examined with a slit lamp and the use of a prototype SOCT instrument constructed in the Institute of Physics, Nicolaus Copernicus University, Torun, Poland. RESULTS: Our SOCT system provides high-resolution (4 microm axial, 10 microm transversal) tomograms composed of 3000-5000 A-scans with an acquisition time of 120-200 ms. The quality of the images is adequate for detailed cross-sectional evaluation of various corneal pathologies. Objective assessment of the localization, size, shape, and light-scattering properties of the changed tissue is possible. Corneal and epithelial thickness and the depth and width of lesions can be estimated. CONCLUSION: SOCT technique allows acquiring clinically valuable cross-sectional optical biopsy of the cornea and its pathologies.

Phase-resolved Doppler optical coherence tomography--limitations and improvements.

We describe how the simple phase difference averaging causes a systematic bias in the velocity estimation obtained by phase-resolved Fourier domain optical coherence tomography (FdOCT). The magnitude of this bias depends on the signal-to-noise ratio as well as proximity of the measured velocity to the limits of the velocity range. We demonstrate the proper way of data processing, which enables obtaining velocity values free of this error. We validate the improved technique by measurements of flow velocity in glass capillaries, in human retinal vessels, and we compare the results with those obtained by standard phase-resolved FdOCT.

Granular corneal dystrophy in 830-nm spectral optical coherence tomography.

PURPOSE: Spectral optical coherence tomography (SOCT) is a new imaging technique that can provide high-resolution tomograms much faster and with higher sensitivity than conventional Time domain (TdOCT) systems. Its usefulness in producing cross-sectional imaging of different corneal pathologies in vivo has already been presented. The aim of this case report is to show 830-nm SOCT findings in granular corneal dystrophy. METHODS: A 48-year-old woman with granular corneal dystrophy was examined with a slit-lamp, confocal microscope (Confoscan 4) and a prototype SOCT instrument constructed at the Institute of Physics, Nicolaus Copernicus University, Torun, Poland. A genetic examination showed a mutation of arginine 555-to-tryptophan (Arg555Trp) in the TGFBI gene that confirmed the clinical diagnosis. RESULTS: SOCT tomograms showed multiple hyperreflective changes throughout the corneal stroma that corresponded to hyaline deposits. Precise and objective assessment of the localization, size, shape, and light scattering properties of the pathologic changes was possible. Three-dimensional rendering of the acquired data allowed a comprehensive evaluation of the deposits in the central cornea. CONCLUSIONS: SOCT (830 nm) provides clinically valuable 2- and 3-dimensional assessments of pathomorphologic changes in granular corneal dystrophy in vivo.

Spectral optical coherence tomography in video-rate and 3D imaging of contact lens wear.

PURPOSE: To present the applicability of spectral optical coherence tomography (SOCT) for video-rate and three-dimensional imaging of a contact lens on the eye surface. METHODS: The SOCT prototype instrument constructed at Nicolaus Copernicus University (Torun, Poland) is based on Fourier domain detection, which enables high sensitivity (96 dB) and increases the speed of imaging 60 times compared with conventional optical coherence tomography techniques. Consequently, video-rate imaging and three-dimensional reconstructions can be achieved, preserving the high quality of the image. The instrument operates under clinical conditions in the Ophthalmology Department (Collegium Medicum Nicolaus Copernicus University, Bydgoszcz, Poland). A total of three eyes fitted with different contact lenses were examined with the aid of the instrument. Before SOCT measurements, slit lamp examinations were performed. RESULTS: Data, which are representative for each imaging mode, are presented. The instrument provided high-resolution (4 microm axial x 10 microm transverse) tomograms with an acquisition time of 40 micros per A-scan. Video-rate imaging allowed the simultaneous quantitative evaluation of the movement of the contact lens and assessment of the fitting relationship between the lens and the ocular surface. Three-dimensional scanning protocols further improved lens visualization and fit evaluation. CONCLUSIONS: SOCT allows video-rate and three-dimensional cross-sectional imaging of the eye fitted with a contact lens. The analysis of both imaging modes suggests the future applicability of this technology to the contact lens field.

Spectral optical coherence tomography: a new imaging technique in contact lens practice.

PURPOSE: Spectral optical coherence tomography (SOCT) is a new non-invasive, non-contact, high-resolution technique, which provides cross-sectional images of objects that weakly absorb and scatter light. The aim of this article is to demonstrate the application of SOCT to imaging of eyes fitted with contact lenses. METHODS: Nine eyes of six different subjects fitted with various contact lenses have been examined with a slit-lamp and a prototype SOCT instrument. RESULTS: Our SOCT system provides high-resolution (4-6 mum longitudinal, 10 mum transversal) tomograms composed of 3000-5000 A-scans with acquisition time of 100-250 ms. The quality of the images is adequate for detailed evaluation of contact lens fit. Design, shape and lens edge position were assessed, and complications of contact lens wear could be visualized. Thickness of the lens, corneal epithelium and stroma as well as the space between the lens and the eye surface have been measured. CONCLUSIONS: SOCT allows high-resolution, cross-sectional visualization of the eye fitted with a contact lens. The ability to carry out a detailed evaluation of the fitting relationship between the lens and the ocular surface might be useful in research and optometric practice. SOCT can also be helpful in diagnosis, evaluation and documentation of contact lens complications.