Spectrally encoded coherence tomography and reflectometry: Simultaneous en face and cross‐sectional imaging at 2 gigapixels per second

Non‐invasive biological imaging is crucial for understanding in vivo structure and function. Optical coherence tomography (OCT) and reflectance confocal microscopy are two of the most widely used optical modalities for exogenous contrast‐free, high‐resolution, three‐dimensional imaging in non‐fluorescent scattering tissues. However, sample motion remains a critical barrier to raster‐scanned acquisition and reconstruction of wide‐field anatomically accurate volumetric datasets. We introduce spectrally encoded coherence tomography and reflectometry (SECTR), a high‐speed, multimodality system for simultaneous OCT and spectrally encoded reflectance (SER) imaging. SECTR utilizes a robust system design consisting of shared optical relays, scanning mirrors, swept laser and digitizer to achieve the fastest reported in vivo multimodal imaging rate of 2 gigapixels per second. Our optical design and acquisition scheme enable spatiotemporally co‐registered acquisition of OCT cross‐sections simultaneously with en face SER images for multivolumetric mosaicking. Complementary axial and lateral translation and rotation are extracted from OCT and SER data, respectively, for full volumetric estimation of sample motion with micron spatial and millisecond temporal resolution.      

MEMS-Based Handheld Fourier Domain Doppler Optical Coherence Tomography for Intraoperative Microvascular Anastomosis Imaging

Purpose

To demonstrate the feasibility of a miniature handheld optical coherence tomography (OCT) imager for real time intraoperative vascular patency evaluation in the setting of super-microsurgical vessel anastomosis.

Methods

A novel handheld imager Fourier domain Doppler optical coherence tomography based on a 1.3-µm central wavelength swept source for extravascular imaging was developed. The imager was minimized through the adoption of a 2.4-mm diameter microelectromechanical systems (MEMS) scanning mirror, additionally a 12.7-mm diameter lens system was designed and combined with the MEMS mirror to achieve a small form factor that optimize functionality as a handheld extravascular OCT imager. To evaluate in-vivo applicability, super-microsurgical vessel anastomosis was performed in a mouse femoral vessel cut and repair model employing conventional interrupted suture technique as well as a novel non-suture cuff technique. Vascular anastomosis patency after clinically successful repair was evaluated using the novel handheld OCT imager.

Results

With an adjustable lateral image field of view up to 1.5 mm by 1.5 mm, high-resolution simultaneous structural and flow imaging of the blood vessels were successfully acquired for BALB/C mouse after orthotopic hind limb transplantation using a non-suture cuff technique and BALB/C mouse after femoral artery anastomosis using a suture technique. We experimentally quantify the axial and lateral resolution of the OCT to be 12.6 µm in air and 17.5 µm respectively. The OCT has a sensitivity of 84 dB and sensitivity roll-off of 5.7 dB/mm over an imaging range of 5 mm. Imaging with a frame rate of 36 Hz for an image size of 1000(lateral)×512(axial) pixels using a 50,000 A-lines per second swept source was achieved. Quantitative vessel lumen patency, lumen narrowing and thrombosis analysis were performed based on acquired structure and Doppler images.

Conclusions

A miniature handheld OCT imager that can be used for intraoperative evaluation of microvascular anastomosis was successfully demonstrated.     

Self‐referenced axial chromatic dispersion measurement in multiphoton microscopy through 2‐color third‐harmonic generation imaging

With tunable excitation light, multiphoton microscopy is widely used for imaging biological structures at subcellular resolution. Axial chromatic dispersion, present in virtually every transmissive optical system including the multiphoton microscope, leads to focal (and the resultant image) plane separation. Here, we experimentally demonstrate a technique to measure the axial chromatic dispersion in a multiphoton microscope, using simultaneous 2‐color third‐harmonic generation imaging excited by a 2‐color soliton source with tunable wavelength separation. Our technique is self‐referenced, eliminating potential measurement error when 1‐color tunable excitation light is used which necessitates reciprocating motion of the mechanical translation stage. Using this technique, we demonstrate measured axial chromatic dispersion with 2 different objective lenses in a multiphoton microscope. Further measurement in a biological sample also indicates that this axial chromatic dispersion, in combination with 2‐color imaging, may open up opportunity for simultaneous imaging of 2 different axial planes.      

Endoscopic optical coherence tomography angiography using a forward imaging piezo scanner probe

A forward imaging endoscope for optical coherence tomography angiography (OCTA) featuring a piezoelectric fiber scanner is presented. Imaging is performed with an optical coherence tomography (OCT) system incorporating an akinetic light source with a center wavelength of 1300 nm, bandwidth of 90 nm and A‐line rate of 173 kHz. The endoscope operates in contact mode to avoid motion artifacts, in particular, beneficial for OCTA measurements, and achieves a transversal resolution of 12 μm in air at a rigid probe size of 4 mm in diameter and 11.3 mm in length. A spiral scan pattern is generated at a scanning frequency of 360 Hz to sample a maximum field of view of 1.3 mm. OCT images of a human finger as well as visualization of microvasculature of the human palm are presented both in two and three dimensions. The combination of morphological tissue contrast with qualitative dynamic blood flow information within this endoscopic imaging approach potentially enables improved early diagnostic capabilities of internal organs for diseases such as bladder cancer.      

Three‐dimensional multiphoton/optical coherence tomography for diagnostic applications in dermatology

A preliminary clinical trial using state‐of‐the‐art multiphoton tomography (MPT) and optical coherence tomography (OCT) for three‐dimensional (3D) multimodal in vivo imaging of normal skin, nevi, scars and pathologic skin lesions has been conducted. MPT enabled visualization of sub‐cellular details with axial and transverse resolutions of <2 μm and <0.5 μm, respectively, from a volume of 0.35 × 0.35 × 0.2 mm³ at a frame rate of 0.14 Hz (512 × 512 pixels). State‐of‐the‐art OCT, operating at a center wavelength of 1300 nm, was capable of acquiring 3D images depicting the layered architecture of skin with axial and transverse resolutions ～8 μm and ～20 μm, respectively, from a volume of 7 × 3.5 × 1.5 mm³ at a frame rate of 46 Hz (1024 × 1024 pixels). This study demonstrates the clinical diagnostic potential of MPT/OCT for pre‐screening relatively large areas of skin using 3D OCT to identify suspicious regions at microscopic level and subsequently using high resolution MPT to obtain zoomed in, sub‐cellular level information of the respective regions (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Multiphoton fluorescence microscopy with GRIN objective aberration correction by low order adaptive optics

Graded Index (GRIN) rod microlenses are increasingly employed in the assembly of optical probes for microendoscopy applications. Confocal, two-photon and optical coherence tomography (OCT) based on GRIN optical probes permit in-vivo imaging with penetration depths into tissue up to the centimeter range. However, insertion of the probe can be complicated by the need of several alignment and focusing mechanisms along the optical path. Furthermore, resolution values are generally not limited by diffraction, but rather by optical aberrations within the endoscope probe and feeding optics. Here we describe a multiphoton confocal fluorescence imaging system equipped with a compact objective that incorporates a GRIN probe and requires no adjustment mechanisms. We minimized the effects of aberrations with optical compensation provided by a low-order electrostatic membrane mirror (EMM) inserted in the optical path of the confocal architecture, resulting in greatly enhanced image quality.     

A compact high‐speed full‐field optical coherence microscope for high‐resolution in vivo skin imaging

A compact high‐speed full‐field optical coherence microscope has been developed for high‐resolution in vivo imaging of biological tissues. The interferometer, in the Linnik configuration, has a size of 11 × 11 × 5 cm³ and a weight of 210 g. Full‐field illumination with low‐coherence light is achieved with a high‐brightness broadband light‐emitting diode. High‐speed full‐field detection is achieved by using part of the image sensor of a high‐dynamic range CMOS camera. En face tomographic images are acquired at a rate of 50 Hz, with an integration time of 0.9 ms. The image spatial resolution is 0.9 μm × 1.2 μm (axial × transverse), over a field of view of 245 × 245 μm². Images of human skin, revealing in‐depth cellular‐level structures, were obtained in vivo and in real‐time without the need for stabilization of the subject. The system can image larger fields, up to 1 × 1 mm², but at a reduced depth.      

Flexible wide‐field optical micro‐angiography based on Fourier‐domain multiplexed dual‐beam swept source optical coherence tomography

Wide‐field optical coherence tomography angiography (OCTA) is gaining interest in clinical imaging applications. In this pursuit, it is challenging to maintain the imaging resolution and sensitivity throughout the wide field of view (FoV). Here, we propose a novel method/system of dual‐beam arrangement and Fourier‐domain multiplexing to achieve wide‐field OCTA when imaging the uneven surface samples. The proposed system provides 2 separate FoVs, with flexibility that the imaging area, focus of the imaging beam and imaging depth range can be individually adjusted for each FoV, leading to either (1) increased system imaging FoV or (2) capability of targeting 2 regions of interests that locate at depths with large difference between each other. We demonstrate this novel method by employing 100 kHz laser source in a swept source OCTA to achieve an effective 200 kHz sweeping rate, covering a 22 × 22 mm FoV. The results are verified by a SS‐OCTA system employing a 200 kHz laser source, together with the experimental demonstrations when imaging whole brain vasculature in rodent models and skin blood perfusion in human fingers, show‐casing the capability of proposed system to image live large samples with complex surface topography.      

Ex vivo investigations on the potential of optical coherence tomography (OCT) as a diagnostic tool for reproductive medicine in a bovine model

Routine infertility investigations in the male and female include imaging techniques such as ultrasonography and endoscopy (fertiloscopy). However, these techniques lack the resolution to localize vital sperm or to reveal detailed morphological analysis of the oviduct which is often the cause of infertility in females. Therefore we set out to evaluate the efficiency of optical coherence tomography (OCT) as a diagnostic imaging tool for micron‐scale visualization of the male and female genital tract. Using the bovine as a model, the optical features of the Telesto^TM, Ganymede^TM (both Thorlabs) and Niris^TM (Imalux) OCT imaging systems were compared.

Comparative visualization of ex vivo bovine testicular tissue by the Telesto^TM microscopic optical coherence tomography system (left) and corresponding H&E staining (right).     

Quantitative and rapid estimations of human sub‐surface skin mass using ultra‐high‐resolution spectral domain optical coherence tomography

Non‐invasive and quantitative estimations for the delineation of sub‐surface tumor margins could greatly aid in the early detection and monitoring of the morphological appearances of tumor growth, ensure complete tumor excision without the unnecessary sacrifice of healthy tissue, and facilitate post‐operative follow‐up for recurrence. In this study, a high‐speed, non‐invasive, and ultra‐high‐resolution spectral domain optical coherence tomography (UHR‐SDOCT) imaging platform was developed for the quantitative measurement of human sub‐surface skin mass. With a proposed robust, semi‐automatic analysis, the system can rapidly quantify lesion area and shape regularity by an en‐face‐oriented algorithm. Various sizes of nylon sutures embedded in pork skin were used first as a phantom to verify the accuracy of our algorithm, and then in vivo, feasibility was proven using benign human angiomas and pigmented nevi. Clinically, this is the first step towards an automated skin lesion measurement system.

In vivo optical coherence tomography (OCT) image of angioma (A). Thin red arrows point to a blood vessel (BV).     

Morphological features of mucous secretory organ and mucous secretion of loach Misgurnus anguillicaudatus skin for friction drag reduction

We examined the functional morphology of loach Misgurnus anguillicaudatus skin by using synchrotron X-ray micro-computed tomography (SR-μCT) and high-contrast staining using osmium tetroxide or phosphotungstic acid (PTA), which enhances the image contrast of soft tissues. The captured high-spatial resolution images revealed that the surface ornamentations were stuck in the basement membrane of the loach scales. The ornamentations consisting of grooves (radii) and ridges (circuli) that can move freely and bend flexibly. The cross-sectional lateral microstructures of flat, concave and convex loach skins were observed from a live image of loach skin obtained through dark-field optical coherence tomography (OCT) imaging. The thickness of loach skin was changed with varying empty space between the mucous-cell layer and the scales by bending motion of loach. In addition, through direct measurement of drag reduction of loach skin, the mucous layer was found to have a strong influence on the reduction of skin friction. The present results enhance the understanding of the functional morphologies of mucous layer of loach to secrete mucus for skin friction reduction.     

Super‐achromatic optical coherence tomography capsule for ultrahigh‐resolution imaging of esophagus

Endoscopic optical coherence tomography (OCT) is a noninvasive technology allowing for imaging of tissue microanatomies of luminal organs in real time. Conventional endoscopic OCT operates at 1300 nm wavelength region with a suboptimal axial resolution limited to 8‐20 μm. In this paper, we present the first ultrahigh‐resolution tethered OCT capsule operating at 800 nm and offering about 3‐ to 4‐fold improvement of axial resolution (plus enhanced imaging contrast). The capsule uses diffractive optics to manage chromatic aberration over a full ~200 nm spectral bandwidth centering around 830 nm, enabling to achieve super‐achromaticity and an axial resolution of ~2.6 μm in air. The performance of the OCT capsule is demonstrated by volumetric imaging of swine esophagus ex vivo and sheep esophagus in vivo, where fine anatomic structures including the sub‐epithelial layers are clearly identified. The ultrahigh resolution and excellent imaging contrast at 800 nm of the tethered capsule suggest the potential of the technology as an enabling tool for surveillance of early esophageal diseases on awake patients without the need for sedation.      

Cell monitoring with optical coherence tomography

Background aimsWe evaluated a commercially available instrument, OCTiCell (chromologic.com/octicell), for monitoring cell growth in suspended agitated bioreactors based on optical coherence tomography. OCTiCell is an in-line, completely non-invasive instrument that can operate on any suspended-cell bioreactor with a window or transparent wall. In traditional optical coherence tomography, the imaging beam is rastered over the sample to form a three-dimensional image. OCTiCell, instead uses a fixed imaging beam and takes advantage of the motion of the media to move the cells across the interrogating optical beam.ResultsWe found strong correlations between the non-invasive, non-contact, reagent-free OCTiCell measurements of cell concentration and viability and those obtained from the automated cell counter, and the XTT viability assay, which is a colorimetric assay for quantifying metabolic activity.ConclusionsThis novel cell monitoring method can adapt to different bioreactor form factors and could reduce the labor cost and contamination risks associated with cell growth monitoring.     

A biopsy‐needle compatible varifocal multiphoton rigid probe for depth‐resolved optical biopsy

In this work, we report a biopsy‐needle compatible rigid probe, capable of performing three‐dimensional (3D) two‐photon optical biopsy. The probe has a small outer diameter of 1.75 mm and fits inside a gauge‐14 biopsy needle to reach internal organs. A carefully designed focus scanning mechanism has been implemented in the rigid probe, which, along with a rapid two‐dimensional MEMS scanner, enables 3D imaging. Fast image acquisition up to 10 frames per second is possible, dramatically reducing motion artifacts during in vivo imaging. Equipped with a high‐numerical aperture micro‐objective, the miniature rigid probe offers a high two‐photon resolution (0.833 × 6.11 μm, lateral × axial), a lateral field of view of 120 μm, and an axial focus tuning range of 200 μm. In addition to imaging of mouse internal organs and subcutaneous tumor in vivo, first‐of‐its‐kind depth‐resolved two‐photon optical biopsy of an internal organ has been successfully demonstrated on mouse kidney in vivo and in situ.      

Quantitative depolarization measurements for fiber‐based polarization‐sensitive optical frequency domain imaging of the retinal pigment epithelium

A full quantitative evaluation of the depolarization of light may serve to assess concentrations of depolarizing particles in the retinal pigment epithelium and to investigate their role in retinal diseases in the human eye. Optical coherence tomography and optical frequency domain imaging use spatial incoherent averaging to compute depolarization. Depolarization depends on accurate measurements of the polarization states at the receiver but also on the polarization state incident upon and within the tissue. Neglecting this dependence can result in artifacts and renders depolarization measurements vulnerable to birefringence in the system and in the sample. In this work, we discuss the challenges associated with using a single input polarization state and traditional depolarization metrics such as the degree‐of‐polarization and depolarization power. We demonstrate quantitative depolarization measurements based on Jones vector synthesis and polar decomposition using fiber‐based polarization‐sensitive optical frequency domain imaging of the retinal pigment epithelium in a human eye.      

Endomicroscopic optical coherence tomography for cellular resolution imaging of gastrointestinal tracts

Our ability to detect neoplastic changes in gastrointestinal (GI) tracts is limited by the lack of an endomicroscopic imaging tool that provides cellular‐level structural details of GI mucosa over a large tissue area. In this article, we report a fiber‐optic‐based micro‐optical coherence tomography (μOCT) system and demonstrate its capability to acquire cellular‐level details of GI tissue through circumferential scanning. The system achieves an axial resolution of 2.48 μm in air and a transverse resolution of 4.8 μm with a depth‐of‐focus (DOF) of ~150 μm. To mitigate the issue of limited DOF, we used a rigid sheath to maintain a circular lumen and center the distal‐end optics. The sensitivity is tested to be 98.8 dB with an illumination power of 15.6 mW on the sample. With fresh swine colon tissues imaged ex vivo, detailed structures such as crypt lumens and goblet cells can be clearly resolved, demonstrating that this fiber‐optic μOCT system is capable of visualizing cellular‐level morphological features. We also demonstrate that time‐lapsed frame averaging and imaging speckle reduction are essential for clearly visualizing cellular‐level details. Further development of a clinically viable μOCT endomicroscope is likely to improve the diagnostic outcome of GI cancers.      

Optical coherence tomography for longitudinal monitoring of vasculature in scars treated with laser fractionation

This study presents the first in vivo longitudinal assessment of scar vasculature in ablative fractional laser treatment using optical coherence tomography (OCT). A method based on OCT speckle decorrelation was developed to visualize and quantify the scar vasculature over the treatment period. Through reliable co‐location of the imaging field of view across multiple imaging sessions, and compensation for motion artifact, the study was able to track the same scar tissue over a period of several months, and quantify changes in the vasculature area density. The results show incidences of occlusion of individual vessels 3 days after the first treatment. The subsequent responses ?20 weeks after the initial treatment show differences between immature and mature scars. Image analysis showed a distinct decrease (25 ± 13%, mean ± standard deviation) and increase (19 ± 5%) of vasculature area density for the immature and mature scars, respectively. This study establishes the feasibility of OCT imaging for quantitative longitudinal monitoring of vasculature in scar treatment.

En face optical coherence tomography vasculature images pre‐treatment (top) and ?20 weeks after the first laser treatment (bottom) of a mature burn scar. Arrows mark the same vessel pattern.     

1064 nm acoustic resolution photoacoustic microscopy

Photoacoustic imaging is a noninvasive imaging technique having the advantages of high‐optical contrast and good acoustic resolution at improved imaging depths. Light transport in biological tissues is mainly characterized by strong optical scattering and absorption. Photoacoustic microscopy is capable of achieving high‐resolution images at greater depth compared to conventional optical microscopy methods. In this work, we have developed a high‐resolution, acoustic resolution photoacoustic microscopy (AR‐PAM) system in the near infra‐red (NIR) window II (NIR‐II, eg, 1064 nm) for deep tissue imaging. Higher imaging depth is achieved as the tissue scattering at 1064 nm is lesser compared to visible or near infrared window‐I (NIR‐I). Our developed system can provide a lateral resolution of 130 μm, axial resolution of 57 μm, and image up to 11 mm deep in biological tissues. This 1064‐AR‐PAM system was used for imaging sentinel lymph node and the lymph vessel in rat. Urinary bladder of rat filled with black ink was also imaged to validate the feasibility of the developed system to study deeply seated organs.      

Comparison of high frequency ultrasound and optical coherence tomography as modalities for high resolution and non invasive skin imaging.

High frequency ultrasound (HFUS) and optical coherence tomography (OCT) are techniques for high resolution imaging of tissues. The penetration depth of these modalities is limited, but it is sufficiently large enough for non invasive skin imaging. HFUS and OCT are based on the same concept. Waves (ultrasonic waves, respectively light waves) propagate along a narrow beam, are backscattered at tissue inhomogeneities and analyzed over time of flight to obtain spatially resolved morphological information. The objective of this paper is to compare HFUS and OCT in terms of resolution, dynamic range and contrast and to assess their value as tools for high resolution skin imaging. Measurements on phantoms and in vivo have been performed with a 100 MHz ultrasound system and an OCT-scanner working in the near infrared spectrum at 1300 nm wave-length. From the measurements, it can be concluded that OCT delivers an almost isotropic resolution (axial resolution about 5.8 microns, lateral resolution about 4.1 microns), whereas the resolution of the investigated HFUS system is more anisotropic (axial resolution about 9.3 microns, lateral resolution about 60 microns). HFUS and OCT show different penetration depths and a different contrast. Both techniques can, therefore, be combined advantageously in a multimodality approach to account for their individual characteristics.     

Interplane bulk motion analysis and removal based on normalized cross‐correlation in optical coherence tomography angiography

Bulk motion seriously degrades the image quality of optical coherence tomography angiography (OCTA). Conventional correction methods focus on in‐plane displacement, while the bulk motion component perpendicular to B‐scans also introduces noise. This work first presents an evaluation of this component using a specific scan protocol and an approximate expression derived from peak‐normalized cross‐correlation values, and then quantitatively assesses how interplane bulk motion noise reduce the sensitivity of cross‐sectional angiograms. Finally, we developed a repetitive bulk motion correction method based on the estimated displacements and redundant volume scans. The correction does not require registration and angiogram reconstruction of low flow sensitivity frames, and the results of in vivo mice skin OCTA imaging experiments show that the proposed method can effectively reduce bulk motion noise caused by cardiac and respiratory motion and occasional shaking, and improve OCTA image quality, which has practical significance for clinical OCTA diagnosis and analysis.