Daniel Szlag

Anterior segment imaging with Spectral OCT system using a high-speed CMOS camera

We describe a new ultrahigh speed Spectral OCT instrument making use of a CMOS camera and demonstrate high quality in vivo imaging of the anterior segment of the human eye. The high flexibility of the designed imaging system allows a wide range of imaging protocols. Two- and three-dimensional high quality OCT images of the cornea, the anterior chamber and the crystalline lens are presented. A high acquisition rate, up to 135,000 A-scans/second enables three-dimensional reconstruction of the anterior segment during lenticular accommodation, blinking and pupillary reaction to light stimulus. We demonstrate OCT tomographic real time imaging of the lens dynamics during accommodation and high quality OCT cross-sectional images of the entire anterior segment of the eye from the cornea up to posterior part of the crystalline lens.

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.

Efficient reduction of speckle noise in Optical Coherence Tomography

Speckle pattern, which is inherent in coherence imaging, influences significantly axial and transversal resolution of Optical Coherence Tomography (OCT) instruments. The well known speckle removal techniques are either sensitive to sample motion, require sophisticated and expensive sample tracking systems, or involve sophisticated numerical procedures. As a result, their applicability to in vivo real-time imaging is limited. In this work, we propose to average multiple A-scans collected in a fully controlled way to reduce the speckle contrast. This procedure involves non-coherent averaging of OCT A-scans acquired from adjacent locations on the sample. The technique exploits scanning protocol with fast beam deflection in the direction perpendicular to lateral dimension of the cross-sectional image. Such scanning protocol reduces the time interval between A-scans to be averaged to the repetition time of the acquisition system. Consequently, the averaging algorithm is immune to bulk motion of an investigated sample, does not require any sophisticated data processing to align cross-sectional images, and allows for precise control of lateral shift of the scanning beam on the object. The technique is tested with standard Spectral OCT system with an extra resonant scanner used for rapid beam deflection in the lateral direction. Ultrahigh speed CMOS camera serves as a detector and acquires 200,000 spectra per second. A dedicated A-scan generation algorithm allows for real-time display of images with reduced speckle contrast at 6 frames/second. This technique is applied to in vivo imaging of anterior and posterior segments of the human eye and human skin.

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.

Quantitative lateral and axial flow imaging with optical coherence microscopy and tomography

Optical coherence tomography (OCT) and optical coherence microscopy (OCM) allow the acquisition of quantitative three-dimensional axial flow by estimating the Doppler shift caused by moving scatterers. Measuring the velocity of red blood cells is currently the principal application of these methods. In many biological tissues, blood flow is often perpendicular to the optical axis, creating the need for a quantitative measurement of lateral flow. Previous work has shown that lateral flow can be measured from the Doppler bandwidth, albeit only for simplified optical systems. In this work, we present a generalized model to analyze the influence of relevant OCT/OCM system parameters such as light source spectrum, numerical aperture and beam geometry on the Doppler spectrum. Our analysis results in a general framework relating the mean and variance of the Doppler frequency to the axial and lateral flow velocity components. Based on this model, we present an optimized acquisition protocol and algorithm to reconstruct quantitative measurements of lateral and axial flow from the Doppler spectrum for any given OCT/OCM system. To validate this approach, Doppler spectrum analysis is employed to quantitatively measure flow in a capillary with both extended focus OCM and OCT.

Complementarity of PALM and SOFI for super-resolution live-cell imaging of focal adhesions.

Live-cell imaging of focal adhesions requires a sufficiently high temporal resolution, which remains a challenge for super-resolution microscopy. Here we address this important issue by combining photoactivated localization microscopy (PALM) with super-resolution optical fluctuation imaging (SOFI). Using simulations and fixed-cell focal adhesion images, we investigate the complementarity between PALM and SOFI in terms of spatial and temporal resolution. This PALM-SOFI framework is used to image focal adhesions in living cells, while obtaining a temporal resolution below 10 s. We visualize the dynamics of focal adhesions, and reveal local mean velocities around 190 nm min-1. The complementarity of PALM and SOFI is assessed in detail with a methodology that integrates a resolution and signal-to-noise metric. This PALM and SOFI concept provides an enlarged quantitative imaging framework, allowing unprecedented functional exploration of focal adhesions through the estimation of molecular parameters such as fluorophore densities and photoactivation or photoswitching kinetics.

Four-dimensional structural and Doppler optical coherence tomography imaging on graphics processing units

Abstract. The authors present the application of graphics processing unit (GPU) programming for real-time three-dimensional (3-D) Fourier domain optical coherence tomography (FdOCT) imaging with implementation of flow visualization algorithms. One of the limitations of FdOCT is data processing time, which is generally longer than data acquisition time. Utilizing additional algorithms, such as Doppler analysis, further increases computation time. The general purpose computing on GPU (GPGPU) has been used successfully for structural OCT imaging, but real-time 3-D imaging of flows has so far not been presented. We have developed software for structural and Doppler OCT processing capable of visualization of two-dimensional (2-D) data (2000 A-scans, 2048 pixels per spectrum) with an image refresh rate higher than 120 Hz. The 3-D imaging of 100×100 A-scans data is performed at a rate of about 9 volumes per second. We describe the software architecture, organization of threads, and optimization. Screen shots recorded during real-time imaging of a flow phantom and the human eye are presented.

Quantitative cerebral blood flow imaging with extended-focus optical coherence microscopy

Quantitative three-dimensional blood flow imaging is a valuable technique to investigate the physiology of the brain. Two-photon microscopy (2PM) allows quantification of the local blood flow velocity with micrometric resolution by performing repeated line scans, but prohibitively long measurement times would be required to apply this technique to full three-dimensional volumes. By multiplexing the image acquisition over depth, Fourier domain optical coherence tomography (FDOCT) enables quantification of blood flow velocities with a high volume acquisition rate, albeit at a relatively low spatial resolution. Extended-focus optical coherence microscopy (xfOCM) increases the lateral resolution without sacrificing depth of field and therefore combines the high volume acquisition rate of FDOCT with a resolution comparable to 2PM. Here, we demonstrate high-resolution quantitative imaging of the blood flow velocity vectors magnitude in the adult murine brain with xfOCM.

Real-time imaging for Spectral Optical Coherence Tomography with massively parallel data processing

The paper presents the way that colour can serve solving the problem of calibration points indexing in a camera geometrical calibration process. We propose a technique in which indexes of calibration points in a black-and-white chessboard are represented as sets of colour regions in the neighbourhood of calibration points. We provide some general rules for designing a colour calibration chessboard and provide a method of calibration image analysis. We show that this approach leads to obtaining better results than in the case of widely used methods employing information about already indexed points to compute indexes. We also report constraints concerning the technique. Nowadays we are witnessing an increasing need for camera geometrical calibration systems. They are vital for such applications as 3D modelling, 3D reconstruction, assembly control systems, etc. Wherever possible, calibration objects placed in the scene are used in a camera geometrical calibration process. This approach significantly increases accuracy of calibration results and makes the calibration data extraction process easier and universal. There are many geometrical camera calibration techniques for a known calibration scene [1]. A great number of them use as an input calibration points which are localised and indexed in the scene. In this paper we propose the technique of calibration points indexing which uses a colour chessboard. The presented technique was developed by solving problems we encountered during experiments with our earlier methods of camera calibration scene analysis [2]-[3]. In particular, the proposed technique increases the number of indexed points points in case of local lack of calibration points detection. At the beginning of the paper we present a way of designing a chessboard pattern. Then we describe a calibration point indexing method, and finally we show experimental results. A black-and-white chessboard is widely used in order to obtain sub-pixel accuracy of calibration points localisation [1]. Calibration points are defined as corners of chessboard squares. Assuming the availability of rough localisation of these points, the points can be indexed. Noting that differences in distances between neighbouring points in calibration scene images differ slightly, one of the local searching methods can be employed (e.g. [2]). Methods of this type search for a calibration point to be indexed, using a window of a certain size. The position of the window is determined by a vector representing the distance between two previously indexed points in the same row or column. However, experiments show that this approach has its disadvantages, as described below. * E-mail: A.Nowakowski@ire.pw.edu.pl Firstly, there is a danger of omitting some points during indexing in case of local lack of calibration points detection in a neighbourhood (e.g. caused by the presence of non-homogeneous light in the calibration scene). A particularly unfavourable situation is when the local lack of detection effects in the appearance of separated regions of detected calibration points. It is worth saying that such situations are likely to happen for calibration points situated near image borders. Such points are very important for the analysis of optical nonlinearities, and a lack of them can significantly influence the accuracy of distortion modelling. Secondly, such methods may give wrong results in the case of optical distortion with strong nonlinearities when getting information about the neighbouring index is not an easy task. Beside this, the methods are very sensitive to a single false localisation of a calibration point. Such a single false localisation can even result in false indexing of a big set of calibration points. To avoid the above-mentioned problems, we propose using a black-and-white chessboard which contains the coded index of a calibration point in the form of colour squares situated in the nearest neighbourhood of each point. The index of a certain calibration point is determined by colours of four nearest neighbouring squares (Fig.1). An order of squares in such foursome is important. Because the size of a colour square is determined only by the possibility of correct colour detection, the size of a colour square can be smaller than the size of a black or white square. The larger size of a black or white square is determined by the requirements of the exact localisation step which follows the indexing of calibration points [3]. In this step, edge information is extracted from a blackand-white chessboard. This edge information needs larger Artur Nowakowski, Wladyslaw Skarbek Institute of Radioelectronics, Warsaw University of Technology, Nowowiejska 15/19, 00-665 Warszawa, A.Nowakowski@ire.pw.edu.pl Received February 10, 2009; accepted March 27, 2009; published March 31, 2009 http://www.photonics.pl/PLP