Coarse-scale Optoretinography (CoORG) is an ORG approach that enables quick (10min), extended-field (5 deg) assessment of retinal structure and function. This study explores the feasibility of CoORG in discerning early and sensitive changes in retinitis pigmentosa (RP). In general, diminished cone function is observed in RP compared to normals using CoORG, even in areas of normal apparent outer retinal structure. This underscores the potential of CoORG for early & sensitive detection of retinal dysfunction in diseases such as RP and AMD.
KEYWORDS: Image registration, Optical coherence tomography, Image segmentation, Visualization, Tissues, Speckle pattern, Signal detection, Motion models, In vivo imaging, Image processing algorithms and systems
Phase-sensitive OCT can be used for imaging the photoreceptor deformations in response to the light stimulus or optoretinography (ORG). Here, we propose a phase-restoring subpixel image registration method and an automated signal extraction algorithm for optoretinography using phase-sensitive OCTs. We validated these methods in simulations, phantom experiments, and in-vivo optoretinogram imaging. Our image registration method yields better amplitude stability and higher phase accuracy compared with conventional approaches, and we found two types of signals (one monophasic and the other biphasic) simultaneously in rodent ORG imaging. These results can be beneficial to the ongoing preclinical/clinical ORG studies.
Optoretinography serves as an effective biomarker for light-evoked retinal activity owing to its sensitive, objective, and precise localization of retinal function and dysfunction. We developed a coarse-scale optoretinography instrument based on a line-scan spectral domain OCT. We demonstrate its ability to acquire robust and repeatable ORG signals rapidly over a 5° field of view in a single acquisition, without adaptive optics. The high repeatability, good agreement with cellular-scale ORGs, and non-AO operation are of promise for its widespread clinical application to retinal diseases such as AMD and inherited retinal degenerations.
Phase-sensitive optical coherence tomography (OCT) enables label-free imaging of structural dynamics with nanoscopic sensitivity. However, the inevitable bulk tissue motions degrade the signal stability and introduce extra phase error. To suppress the motion-induced phase error, we propose a phase-restoring subpixel motion correction method for post-hoc motion correction in Fourier domain OCT, which enables translational shifts of complex-valued OCT images by arbitrary distance. Phantom and in-vivo rodent optoretinogram imaging experiments were conducted to demonstrate the advantages of the proposed method over conventional pixel-level method and the Fourier transform based method.
We introduce reflective mirror-based line-scan adaptive optics line-scan OCT, optimized for imaging light-induced retinal activity (optoretinography) and weak retinal reflections at the cellular scale. The performance was exemplified by cellular-scale visualization of retinal ganglion cells, macrophages, foveal cones, and rods in human observers. Light-evoked optical changes in foveal cones were observable at an eccentricity 0.3 deg. from the foveal center, enabling the first in vivo demonstration of reduced S-cone (short-wavelength cone) density in the human foveola. Given the challenges typically associated with optical accessibility in the living human fovea, this instrument holds significant promise for basic and translational applications.
We present a method for determining the optical and thermal properties of layered materials, applicable to retinal laser therapy, using phase-resolved OCT. Transient heating of a tissue phantom is achieved by focusing a laser pulse onto a buried absorbing layer. Optical path length changes between the top of the phantom and the scattering absorbing layer induced by material expansion are extracted from the sequential B-scans. The absorption coefficient, heat conductivity and thermal expansion coefficient of the polymer are determined by matching the experimental data to a thermomechanical model of the tissue, yielding a temperature precision <2%, well below damage threshold.
We present a methodology that allows precise determination of the optical and thermal properties of layered materials using thermal perturbation and phase-resolved optical coherence tomography (pOCT). Following heating with a laser pulse of a buried absorbing layer, optical path length changes between the sample’s surface and the absorbing layer are monitored using a line-scan pOCT. Using an axisymmetric thermo-mechanical model for transversely uniform multi-layered media, we fit the absorption coefficient, the heat conductivity, and the thermal expansion coefficient of the polymer. We demonstrate that the temperature distribution can be determined with a precision under 0.1°C, after a single laser pulse.
We introduce an anamorphic detection paradigm to optimize spatial and spectral resolution in adaptive optics line-scan OCT, wherein an improved light collection efficiency and signal roll-off compared to traditional methods was demonstrated. The benefits for in vivo imaging were exemplified by retrieving nanometer-scale light-induced optical path length dynamics at high speed in individual cones. The high speed, sensitivity and cellular-scale resolution of the resulting adaptive optics line-scan OCT instrument offers a robust and sensitive biomarker for retinal function in health and disease.
We developed a high-speed adaptive optics, line-scan spectral domain OCT and used it to characterize stimulus-induced optical path length changes in cones with high spatiotemporal resolution. We find that individual cone outer segments exhibit a biphasic light-induced response—a rapid axial shrinkage followed by a gradual increase in optical path length, both increasing in magnitude with the stimulus intensity. AO line-scan OCT thus offers high-speed volume acquisitions, high phase stability, sub-ms temporal resolution and cellular-scale spatial resolution, that together enable imaging retinal structure and function in health and disease.
Conventional cytology is a rapid chair-side method for diagnosis, but it relies on laborious fixing and staining protocols. As cytology specimens are transparent, it is very hard to visualize them under a bright-field microscope without staining. Quantitative phase imaging techniques have opened up an interesting and potential diagnostic method for volumetric three-dimensional (3-D) visualization of the transparent specimens without any need for sample preparations. We explore the use of digital holographic microscopy in clinical application of oral cytology for the 3-D visualization of buccal cells with high contrast without any additional sample preparations. We also propose nuclear to cytoplasmic (N / C) volumes as a much more accurate parameter for identification of multinucleate and actively dividing cells. We quantify the cellular volumes, and N / C ratios for 203 buccal cells taken from five healthy volunteers to clinically validate the technique and compare them with the traditional N / C area ratios as well as the histology standards. The mean N / C area and volume ratios are found to be 0.0322 ± 0.0149 and 0.0648 ± 0.0286, respectively. Our approach highlights the dawn of a new method for a label-free/nondestructive volumetric oral cytology evaluation, with high potential for exploration of suspicious oral lesions, in subjects with chronic habits such as alcoholism and tobacco use.
A constrained optimization approach with faster convergence is proposed to recover the complex object field from a near on-axis digital holography (DH). We subtract the DC from the hologram after recording the object beam and reference beam intensities separately. The DC-subtracted hologram is used to recover the complex object information using a constrained optimization approach with faster convergence. The recovered complex object field is back propagated to the image plane using the Fresnel back-propagation method. The results reported in this approach provide high-resolution images compared with the conventional Fourier filtering approach and is 25% faster than the previously reported constrained optimization approach due to the subtraction of two DC terms in the cost function. We report this approach in DH and digital holographic microscopy using the U.S. Air Force resolution target as the object to retrieve the high-resolution image without DC and twin image interference. We also demonstrate the high potential of this technique in transparent microelectrode patterned on indium tin oxide-coated glass, by reconstructing a high-resolution quantitative phase microscope image. We also demonstrate this technique by imaging yeast cells.
We demonstrate a single-shot near on-axis digital holographic microscope that uses a constrained optimization approach for retrieval of the complex object function in the hologram plane. The recovered complex object is back-propagated from the hologram plane to image plane using the Fresnel back-propagation algorithm. A numerical aberration compensation algorithm is employed for correcting the aberrations in the object beam. The reference beam angle is calculated automatically using the modulation property of Fourier transform without any additional recording. We demonstrate this approach using a United States Air Force (USAF) resolution target as an object on our digital holographic microscope. We also demonstrate this approach by recovering the quantitative phase images of live yeast cells, red blood cells and dynamics of live dividing yeast cells.
Most living cells are optically transparent which makes it difficult to visualize them under bright field microscopy. Use of contrast agents or markers and staining procedures are often followed to observe these cells. However, most of these staining agents are toxic and not applicable for live cell imaging. In the last decade, quantitative phase imaging has become an indispensable tool for morphological characterization of the phase objects without any markers. In this paper, we report noninterferometric quantitative phase imaging of live sperm cells by solving transport of intensity equations with recorded intensity measurements along optical axis on a commercial bright field microscope.
Quantitative phase imaging (QPI) aims at studying weakly scattering and absorbing biological specimens with subwavelength accuracy without any external staining mechanisms. Use of a reference beam at an angle is one of the necessary criteria for recording of high resolution holograms in most of the interferometric methods used for quantitative phase imaging. The spatial separation of the dc and twin images is decided by the reference beam angle and Fourier-filtered reconstructed image will have a very poor resolution if hologram is recorded below a minimum reference angle condition. However, it is always inconvenient to have a large reference beam angle while performing high resolution microscopy of live cells and biological specimens with nanometric features. In this paper, we treat reconstruction of digital holographic microscopy images as a constrained optimization problem with smoothness constraint in order to recover only complex object field in hologram plane even with overlapping dc and twin image terms. We solve this optimization problem by gradient descent approach iteratively and the smoothness constraint is implemented by spatial averaging with appropriate size. This approach will give excellent high resolution image recovery compared to Fourier filtering while keeping a very small reference angle. We demonstrate this approach on digital holographic microscopy of live cells by recovering the quantitative phase of live cells from a hologram recorded with nearly zero reference angle.
Digital holographic microscope (DHM) is an emerging quantitative phase imaging technique with unique imaging scales and resolutions leading to multitude of applications. DHM is promising as a novel investigational and applied tool for cell imaging, studying the morphology and real time dynamics of cells and a number of related applications. The use of numerical propagation and computational digital optics offer unique flexibility to tune the depth of focus, and compensate for image aberrations. In this work, we report imaging the dynamics of cell division in E.coli and yeast cells using a DHM platform. We demonstrate 3-D and depth imaging as well as reconstruction of phase profiles of E.coli and yeast cells using the system. We record a digital hologram of E.coli and yeast cells and reconstruct the image using Fresnel propagation algorithm. We also use aberration compensation algorithms for correcting the aberrations that are introduced by the microscope objective in the object path using linear least square fitting techniques. This work demonstrates the strong potential of a DHM platform in 3-D live cell imaging, fast clinical quantifications and pathological applications.
Real-time imaging of live cells is quite difficult without the addition of external contrast agents. Various methods for quantitative phase imaging of living cells have been proposed like digital holographic microscopy and diffraction phase microscopy. In this paper, we report theoretical and experimental results of quantitative phase imaging of live yeast cells with nanometric precision using transport of intensity equations (TIE). We demonstrate nanometric depth sensitivity in imaging live yeast cells using this technique. This technique being noninterferometric, does not need any coherent light sources and images can be captured through a regular bright-field microscope. This real-time imaging technique would deliver the depth or 3-D volume information of cells and is highly promising in real-time digital pathology applications, screening of pathogens and staging of diseases like malaria as it does not need any preprocessing of samples.
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