Phase-resolved Optical Coherence Tomography Research Articles

Light-evoked deformations in rod photoreceptors, pigment epithelium and subretinal space revealed by prolonged and multilayered optoretinography

Phototransduction involves changes in concentration of ions and other solutes within photoreceptors and in subretinal space, which affect osmotic pressure and the associated water flow. Corresponding expansion and contraction of cellular layers can be imaged using optoretinography (ORG), based on phase-resolved optical coherence tomography (OCT). Until now, ORG could reliably detect only photoisomerization and phototransduction in photoreceptors, primarily in cones under bright stimuli. Here, by employing a phase-restoring subpixel motion correction algorithm, which enables imaging of the nanometer-scale tissue dynamics during minute-long recordings, and unsupervised learning of spatiotemporal patterns, we discover optical signatures of the other retinal structures’ response to visual stimuli. These include inner and outer segments of rod photoreceptors, retinal pigment epithelium, and subretinal space in general. The high sensitivity of our technique enables detection of the retinal responses to dim stimuli: down to 0.01% bleach level, corresponding to natural levels of scotopic illumination. We also demonstrate that with a single flash, the optoretinogram can map retinal responses across a 12° field of view, potentially replacing multifocal electroretinography. This technique expands the diagnostic capabilities and practical applicability of optoretinography, providing an alternative to electroretinography, while combining structural and functional retinal imaging in the same OCT machine.

Interferometric thermometry of ocular tissues for retinal laser therapy.

Controlling the tissue temperature rise during retinal laser therapy is highly desirable for predictable and reproducible outcomes of the procedure, especially with non-damaging settings. In this work, we demonstrate a method for determining the optical absorption, the thermal conductivity, and the thermal expansion coefficients of RPE and choroid using phase-resolved optical coherence tomography (pOCT). These parameters are extracted from the measured changes in the optical path length (ΔOPL) using an axisymmetric thermo-mechanical model. This allows the calculation of the temperature rise during hyperthermia, which was further validated by imaging the temperature-sensitive fluorescence at the same location. We demonstrate that, with a temperature uncertainty of ±0.9°C and a peak heating of about 17°C following a laser pulse of 20 ms, this methodology is expected to be safe and sufficiently precise for calibration of the non-damaging retinal laser therapy. The method is directly translatable to in-vivo studies, where we expect a similar precision.

Human cone elongation responses can be explained by photoactivated cone opsin and membrane swelling and osmotic response to phosphate produced by RGS9-catalyzed GTPase

Human cone outer segment (COS) length changes in response to stimuli bleaching up to 99% of L- and M-cone opsins were measured with high resolution, phase-resolved optical coherence tomography (OCT). Responses comprised a fast phase (∼5 ms), during which COSs shrink, and two slower phases (1.5 s), during which COSs elongate. The slower components saturated in amplitude (∼425 nm) and initial rate (∼3 nm ms-1) and are well described over the 200-fold bleaching range as the sum of two exponentially rising functions with time constants of 80 to 90 ms (component 1) and 1,000 to 1,250 ms (component 2). Measurements with adaptive optics reflection densitometry revealed component 2 to be linearly related to cone pigment bleaching, and the hypothesis is proposed that it arises from cone opsin and disk membrane swelling triggered by isomerization and rate-limited by chromophore hydrolysis and its reduction to membrane-localized all-trans retinol. The light sensitivity and kinetics of component 1 suggested that the underlying mechanism is an osmotic response to an amplified soluble by-product of phototransduction. The hypotheses that component 1 corresponds to G-protein subunits dissociating from the membrane, metabolites of cyclic guanosine monophosphate (cGMP) hydrolysis, or by-products of activated guanylate cyclase are rejected, while the hypothesis that it corresponds to phosphate produced by regulator of G-protein signaling 9 (RGS9)-catalyzed hydrolysis of guanosine triphosphate (GTP) in G protein-phosphodiesterase complexes was found to be consistent with the results. These results provide a basis for the assessment with optoretinography of phototransduction in individual cone photoreceptors in health and during disease progression and therapeutic interventions.

Interferometric imaging of thermal expansion for temperature control in retinal laser therapy.

Precise control of the temperature rise is a prerequisite for proper photothermal therapy. In retinal laser therapy, the heat deposition is primarily governed by the melanin concentration, which can significantly vary across the retina and from patient to patient. In this work, we present a method for determining the optical and thermal properties of layered materials, directly applicable to the retina, using low-energy laser heating and phase-resolved optical coherence tomography (pOCT). The method is demonstrated on a polymer-based tissue phantom heated with a laser pulse focused onto an absorbing layer buried below the phantom's surface. Using a line-scan spectral-domain pOCT, optical path length changes induced by the thermal expansion were extracted from sequential B-scans. The material properties were then determined by matching the optical path length changes to a thermo-mechanical model developed for fast computation. This method determined the absorption coefficient with a precision of 2.5% and the temperature rise with a precision of about 0.2°C from a single laser exposure, while the peak did not exceed 8°C during 1 ms pulse, which is well within the tissue safety range and significantly more precise than other methods.

Mechanisms of Light-Induced Deformations in Photoreceptors

Biological cells deform on a nanometer scale when their transmembrane voltage changes, an effect that has been visualized during the action potential using quantitative phase imaging. Similar changes in the optical path length have been observed in photoreceptor outer segments after a flash stimulus via phase-resolved optical coherence tomography. These optoretinograms reveal a fast, millisecond-scale contraction of the outer segments by tens of nanometers, followed by a slow (hundreds of milliseconds) elongation reaching hundreds of nanometers. Ultrafast measurements of the contractile response using line-field phase-resolved optical coherence tomography show a logarithmic increase in amplitude and a decreasing time to peak with increasing stimulus intensity. We present a model that relates the early receptor potential to these deformations based on the voltage-dependent membrane tension—the mechanism observed earlier in neurons and other electrogenic cells. The early receptor potential is caused by conformational changes in opsins after photoisomerization, resulting in the fractional shift of the charge across the disk membrane. Lateral repulsion of the ions on both sides of the membrane affects its surface tension and leads to its lateral expansion. Because the volume of the disks does not change on a millisecond timescale, their lateral expansion leads to an axial contraction of the outer segment. With increasing stimulus intensity and the resulting tension, the area expansion coefficient of the disk membrane also increases as thermally induced fluctuations are pulled flat, resisting further expansion. This leads to the logarithmic saturation observed in measurements as well as the peak shift in time. This imaging technique therefore relates the structural changes in the photoreceptor to the underlying neurological function of transducing light into electrical signals. Such label-free optical monitoring of neural activity using fast interferometry may be applicable not only to optoretinography but also to neuroscience in general.

Assessment of corneal viscoelasticity using elastic wave optical coherence elastography.

The corneal viscoelasticity have great clinical significance, such as the early diagnosis of keratoconus. In this work, an analysis method which utilized the elastic wave velocity, frequency and energy attenuation to assess the corneal viscoelasticity is presented. Using phase-resolved optical coherence tomography, the spatial-temporal displacement map is derived. The phase velocity dispersion curve and center frequency are obtained by transforming the displacement map into the wavenumber-frequency domain through the 2D fast Fourier transform (FFT). The shear modulus is calculated through Rayleigh wave equation using the phase velocity in the high frequency. The normalized energy distribution is plotted by transforming the displacement map into the spatial-frequency domain through the 1D FFT. The energy attenuation coefficient is derived by exponential fitting to calculate the viscous modulus. Different concentrations of tissue-mimicking phantoms and porcine corneas are imaged to validate this method, which demonstrates that the method has the capability to assess the corneal viscoelasticity.

Interferometric mapping of material properties using thermal perturbation

Optical phase changes induced by transient perturbations provide a sensitive measure of material properties. We demonstrate the high sensitivity and speed of such methods, using two interferometric techniques: quantitative phase imaging (QPI) in transmission and phase-resolved optical coherence tomography (OCT) in reflection. Shot-noise-limited QPI can resolve energy deposition of about 3.4 mJ/cm2 in a single pulse, which corresponds to 0.8 °C temperature rise in a single cell. OCT can detect deposition of 24 mJ/cm2 energy between two scattering interfaces producing signals with about 30-dB signal-to-noise ratio (SNR), and 4.7 mJ/cm2 when SNR is 45 dB. Both techniques can image thermal changes within the thermal confinement time, which enables accurate single-shot mapping of absorption coefficients even in highly scattering samples, as well as electrical conductivity and many other material properties in biological samples at cellular scale. Integration of the phase changes along the beam path helps increase sensitivity, and the signal relaxation time reveals the size of hidden objects. These methods may enable multiple applications, ranging from temperature-controlled retinal laser therapy or gene expression to mapping electric current density and characterization of semiconductor devices with rapid pump-probe measurements.

Capillary red blood cell velocimetry by phase-resolved optical coherence tomography.

We present a phase-resolved optical coherence tomography (OCT) method to extend Doppler OCT for the accurate measurement of the red blood cell (RBC) velocity in cerebral capillaries. OCT data were acquired with an M-mode scanning strategy (repeated A-scans) to account for the single-file passage of RBCs in a capillary, which were then high-pass filtered to remove the stationary component of the signal to ensure an accurate measurement of phase shift of flowing RBCs. The angular frequency of the signal from flowing RBCs was then quantified from the dynamic component of the signal and used to calculate the axial speed of flowing RBCs in capillaries. We validated our measurement by RBC passage velocimetry using the signal magnitude of the same OCT time series data.

Detection of local tissue alteration during retinal laser photocoagulation of ex vivo porcine eyes using phase-resolved optical coherence tomography.

Retinal laser photocoagulation is used to treat several ophthalmic diseases. However, it is associated with damage to surrounding healthy tissue. Local tissue alteration during coagulation laser illumination was measured using phase-resolved optical coherence tomography (OCT) M-mode scan as a change in the local optical path length (LOPL). A metric that represents global net tissue alteration was defined using the LOPL change. The visibility of a laser lesion was assessed by three-dimensional OCT volume measurement. Multiple logistic regression analysis was performed to investigate the association between the introduced metric and the laser lesion visibility. The metric was found to be a statistically significant predictor of the laser lesion visibility independent to laser condition. The proposed method based on an LOPL change is thus promising for retinal photocoagulation monitoring.

Nanoscale surface topography imaging using phase-resolved spectral domain optical coherence tomography

Microscopic surface topography plays an important role in studying the functions and properties of materials. Microscopic surface topography measurement has been widely used in many areas, such as machine manufacturing, electronic industry and biotechnology. Optical interferometry is a popular technique for surface topography measurement with an axial resolution up to nanoscale. However, the application of this technique is hampered by phase wrapping, which results in a limited measurement range for this technique. Various digital algorithms for phase unwrapping have been proposed based on the phase continuity between two adjacent points. However, several significant challenges still exist in recovering correct phase with this technique. Optical coherence tomography (OCT) is a non-contact three-dimensional imaging modality with high spatial resolution, and it has been widely used for imaging the biological tissues. In this paper, we demonstrate a method for nanoscale imaging of surface topography by using common-path phase-resolved spectral domain OCT to reduce the influence of phase wrapping. The system includes a superluminescent diode with a central wavelength of 1310 nm and a spectral bandwidth of 62 nm, an optical fiber circulator, a home-made spectrometer, and a reference arm and a sample arm in common-path arrangement. The reference mirror and the sample under investigation are positioned on a same stage in order to further reduce the influence of ambient vibration. The phase difference between two adjacent points is calculated by performing Fourier transform on the measured interferometric spectrum. The phase difference distribution of the surface is obtained first. And then, the surface topography of the sample is constructed by integrating the phase difference distribution. In the traditional methods, phase wrapping occurs if the absolute value of the measured phase is greater than . However, in the present method, phase wrapping occurs if the absolute value of the phase difference between two adjacent points is greater than . The maximal detectable absolute value of the phase difference between two adjacent points increases from for the traditional methods to 2 for the present method. The experimental results indicate that the present system has a high stability and the maximum fluctuation is less than 0.3 nm without averaging. The accuracy of the system is tested with a piezo stage, and the mean absolute deviation of the measured results is 0.62 nm. The performance of the present system is also demonstrated by the surface topography imaging of an optical resolution test target and a roughness comparison specimen. The experimental result shows that the present system is a potential powerful tool for surface topography imaging with an axial resolution better than 1 nm.

In vivo photothermal optical coherence tomography for non-invasive imaging of endogenous absorption agents

In vivo photothermal optical coherence tomography (OCT) is demonstrated for cross-sectional imaging of endogenous absorption agents. In order to compromise the sensitivity, imaging speed, and sample motion immunity, a new photothermal detection scheme and phase processing method are developed. Phase-resolved swept-source OCT and fiber-pigtailed laser diode (providing excitation at 406 nm) are combined to construct a high-sensitivity photothermal OCT system. OCT probe and excitation beam coaxially illuminate and are focused on tissues. The photothermal excitation and detection procedure is designed to obtain high efficiency of photothermal effect measurement. The principle and method of depth-resolved cross-sectional imaging of absorption agents with photothermal OCT has been derived. The phase-resolved thermal expansion detection algorithm without motion artifact enables in vivo detection of photothermal effect. Phantom imaging with a blood phantom and in vivo human skin imaging are conducted. A phantom with guinea-pig blood as absorber has been scanned by the photothermal OCT system to prove the concept of cross-sectional absorption agent imaging. An in vivo human skin measurement is also performed with endogenous absorption agents.

Noise statistics of phase-resolved optical coherence tomography imaging: single-and dual-beam-scan Doppler optical coherence tomography

Noise statistics of phase-resolved optical coherence tomography (OCT) imaging are complicated and involve noises of OCT, correlation of signals, and speckles. In this paper, the statistical properties of phase shift between two OCT signals that contain additive random noises and speckle noises are presented. Experimental results obtained with a scattering tissue phantom are in good agreement with theoretical predictions. The performances of the dual-beam method and conventional single-beam method are compared. As expected, phase shift noise in the case of the dual-beam-scan method is less than that for the single-beam method when the transversal sampling step is large.

Imaging the electro-kinetic response of biological tissues with phase-resolved optical coherence tomography

AbstractIn this study, the electro-kinetic phenomena (EKP) induced in biological tissue by external electric field, while not directly visible in optical coherence tomography (OCT) images, were detected by analyzing their textural speckle features. During application of a low-frequency electric field to the tissue, speckle patterns changed their brightness and shape depending on the local tissue EKP. Since intensities of OCT image speckle patterns were analyzed and discussed in our previous publications, this work is mainly focused on OCT signal phase analysis. The algorithm for extracting local spatial phase variations from unwrapped phases is introduced. The detection of electrically induced optical changes manifest in OCT phase images shows promise for monitoring the fixed charge density changes within tissues through their electro-kinetic responses. This approach may help in the identification and characterization of morphology and function of healthy and pathologic tissues.

Imaging and quantifying Brownian motion of micro- and nanoparticles using phase-resolved Doppler variance optical coherence tomography

Different types and sizes of micro- and nanoparticles have been synthesized and developed for numerous applications. It is crucial to characterize the particle sizes. Traditional dynamic light scattering, a predominant method used to characterize particle size, is unable to provide depth resolved information or imaging functions. Doppler variance optical coherence tomography (OCT) measures the spectral bandwidth of the Doppler frequency shift due to the Brownian motion of the particles utilizing the phase-resolved approach and can provide quantitative information about particle size. Spectral bandwidths of Doppler frequency shifts for various sized particles were quantified and were demonstrated to be inversely proportional to the diameter of the particles. The study demonstrates the phase-resolved Doppler variance spectral domain OCT technique has the potential to be used to investigate the properties of particles in highly scattering media.

Stripe motion artifact suppression in phase-resolved OCT blood flow images of the human eye based on the frequency rejection filter

Stripe motion artifacts caused by phase fluctuation in phase-resolved optical coherence tomography (OCT) result in the quality degradation of the image ofin vivo blood flow of human eye. In order to suppress the stripe motion artifacts, we design a kind of frequency rejection filter aimed at the frequency spectrum characteristics of the image. Blood flow images of human eye acquired by our research group and another group are filtered to show the performance of the proposed method. Experimental results indicate that the stripe motion artifacts in the projection images are rejected significantly with minimal loss of signal information. The proposed filter can also be used in other imaging systems with similar stripe noise.

Phase-resolved acoustic radiation force optical coherence elastography

Many diseases involve changes in the biomechanical properties of tissue, and there is a close correlation between tissue elasticity and pathology. We report on the development of a phase-resolved acoustic radiation force optical coherence elastography method (ARF-OCE) to evaluate the elastic properties of tissue. This method utilizes chirped acoustic radiation force to produce excitation along the sample's axial direction, and it uses phase-resolved optical coherence tomography (OCT) to measure the vibration of the sample. Under 500-Hz square wave modulated ARF signal excitation, phase change maps of tissue mimicking phantoms are generated by the ARF-OCE method, and the resulting Young's modulus ratio is correlated with a standard compression test. The results verify that this technique could efficiently measure sample elastic properties accurately and quantitatively. Furthermore, a three-dimensional ARF-OCE image of the human atherosclerotic coronary artery is obtained. The result indicates that our dynamic phase-resolved ARF-OCE method can delineate tissues with different mechanical properties.

Motion correction for phase-resolved dynamic optical coherence tomography imaging of rodent cerebral cortex

Cardiac and respiratory motions in animals are the primary source of image quality degradation in dynamic imaging studies, especially when using phase-resolved imaging modalities such as spectral-domain optical coherence tomography (SD-OCT), whose phase signal is very sensitive to movements of the sample. This study demonstrates a method with which to compensate for motion artifacts in dynamic SD-OCT imaging of the rodent cerebral cortex. We observed that respiratory and cardiac motions mainly caused, respectively, bulk image shifts (BISs) and global phase fluctuations (GPFs). A cross-correlation maximization-based shift correction algorithm was effective in suppressing BISs, while GPFs were significantly reduced by removing axial and lateral global phase variations. In addition, a non-origin-centered GPF correction algorithm was examined. Several combinations of these algorithms were tested to find an optimized approach that improved image stability from 0.5 to 0.8 in terms of the cross-correlation over 4 s of dynamic imaging, and reduced phase noise by two orders of magnitude in ~8% voxels.

Determination of suspension viscosity from the flow velocity profile measured by Doppler Optical Coherence Tomography

In this paper we present, for the first time to our knowledge, the experimental results for determining suspension viscosity in a capillary type viscometer from flow velocity profiles measured by Doppler Optical Coherence Tomography. The suspension of 0.3?m polystyrene microspheres in a glycerol-water solution was used as a model fluid. The viscosity of the suspension was controlled by glycerol concentration. The shear rate and the shear stress at the capillary wall were measured at six flow rates. The corresponding viscosities were calculated. The comparison of the measured shear rates with the values calculated according to the flow rate of the syringe precision pump was performed. Additionally, the viscosities of the studied suspensions were measured by a rotational viscometer. Full Text: PDFReferences K.A. Hayes, M.R. buckley, I. Cohen, L.A. Archer, "High Resolution Shear Profile Measurements in Entangled Polymers", Phys. Rev. Lett. 101, 218301 (2008)[CrossRef] N. Willenbacher, H. Hanciogullari, H.G. Wagner, "High shear rheology of paper coating colors ? more than just viscosity", Chem. Eng. Technol. 20, 557 (1997)[CrossRef] M. Iotti, O.W. Gregersen, S. Moe, M. Lenes, "Rheological Studies of Microfibrillar Cellulose Water Dispersions", J. Polym. Environ. 19, 137 (2011)[CrossRef] M.T. Roberts, A. Mohraz, K.T. Christensen, J.A. Lewis, "Direct Flow Visualization of Colloidal Gels in Microfluidic Channels", Langmuir 23, 8726 (2007)[CrossRef] M. Schmidt, E. Wassner, H. Münstedt, "Setup and Test of a Laser Doppler Velocimeter for Investigations of Flow Behaviour of Polymer Melts", Mech. Time-Depend. Mater. 3, 371, (1999)[CrossRef] S. Schuberth, H. Münstedt, "Simultaneous measurements of velocity and stress distributions in polyisobutylenes using laser-Doppler velocimetry and flow induced birefringence", Rheol. Acta 47, 111 (2008)[CrossRef] Th. Wunderlich, P.O. Brunn, "A wall layer correction for ultrasound measurement in tube flow: comparison between theory and experiment", Flow Meas. Instrum. 11, 63 (2000)[CrossRef] J. Wiklund, I. Shahram, M. Stading, "Methodology for in-line rheology by ultrasound Doppler velocity profiling and pressure difference techniques", Chem. Eng. Sci. 62, 4277 (2007)[CrossRef] S. Yazdanfar, M.D. Kulkarni, J.A. Izatt, "High resolution imaging of in vivo cardiac dynamics using color Doppler optical coherence tomography", Opt. Express 1(13), 424 (1997)[CrossRef] Y. Zhao et al., "Phase-resolved optical coherence tomography and optical Doppler tomography for imaging blood flow in human skin with fast scanning speed and high velocity sensitivity", Opt. Lett. 25(2), 114 (2000)[CrossRef] J. Moger, S.J. Matcher C.P. Winlove, A. Shore, "Measuring red blood cell flow dynamics in a glass capillary using Doppler optical coherence tomography and Doppler amplitude optical coherence tomography", J. Biomed. Opt. 9(5), 982 (2004)[CrossRef] S. Tamborski et al., "Simultaneous analysis of flow velocity and spectroscopic properties of scattering media with the use of joint Spectral and Time domain OCT", Phot. Lett. Poland 1(2), 49 (2009) B. Povazay et al., "Submicrometer axial resolution optical coherence tomography", Opt. Lett. 27(20), 1800 (2002)[CrossRef] M. Harvey, T.A. Waigh, "Optical coherence tomography velocimetry in controlled shear flow", Phys. Rev. E 83, 031502 (2011)[CrossRef] J. Lauri, M. Wang, M. Kinnunen, R. Myllylä, "Measurement of microfluidic flow velocity profile with two Doppler optical coherence tomography systems", Proc. SPIE 6863, 68630F (2008)[CrossRef]

PHASE DETECTION WITH SUB-NANOMETER SENSITIVITY USING POLARIZATION QUADRATURE ENCODING METHOD IN OPTICAL COHERENCE TOMOGRAPHY

This paper presents a phase-resolved optical coherence tomography (OCT) system that uses the polarization quadrature encoding method in a two-channel Mach-Zehnder interferometer. OCT is a powerful optical signal acquisition method that can capture depth-resolved micrometer-resolution images. In our method, a complex signal is optically generated, and its real and imaginary components are encoded in the orthogonal polarization states of one sample beam; absolute phase information can then be acquired instantaneously. Neither phase modulation nor numerical Fourier or Hilbert transformation to extract phase information is required, thereby decreasing data acquisition rates and processing time. We conducted signal post-processing to select data from the instabilities of reference scanning delay lines; the measured phase sensitivity was as low as 0.23 - , and the corresponding path-difierence resolution was 265pm. A localized surface proflle measurement of a chromium- coated layer deposited on a commercial resolution target surface was conducted. The results conflrmed that successful images can be obtained even with very small optical path difierences using the proposed method.

Phase-resolved magnetomotive OCT for imaging nanomolar concentrations of magnetic nanoparticles in tissues

Magnetic nanoparticles (MNPs) are increasingly important in magnetic resonance and biomedical optical imaging. We describe a method for imaging MNPs by detecting nanoscale displacements using a phase-resolved spectral-domain optical coherence tomography (OCT) system. Biological tissues and phantoms are exposed to approximately 800 G magnetic fields modulated at 56 and 100 Hz to mechanically actuate embedded iron oxide MNPs (approximately 20 nm diameter). Sensitivity to 27 microg/g (approximately 2 nM) MNPs within tissue phantoms is achieved by filtering paramagnetic from diamagnetic vibrations. We demonstrate biological feasibility by imaging topically applied MNPs during their diffusion into an excised rat tumor over a 2 hour time period.