Μm Axial Resolution Research Articles

Video-rate two-photon microendoscopy using second harmonic resonance fiber scanning

We present a 2.5-mm-diameter resonant fiber scanning two-photon microendoscope with a 30-mm long forward-viewing rigid probe tip that enables video-rate imaging (20 Hz frame rate) suitable for hand-held imaging of tissues without motion artifacts. Higher-order harmonic oscillation scanning techniques are developed to significantly increase the frame rate compared to prior published fiber scanning microendoscopy designs while maintaining the field-of-view (∼125 µm), the optical resolution (1.2 µm lateral and 10.9 µm axial resolution, full width at half maximum), and the spatial sampling (1250 circumferential pixels per spiral × 20 radial pixels over the diameter; 210 spirals per frame, ∼4 spiral samples per resolvable pixel) compared to a traditional scan using the fundamental resonance. 3D printed mounts were created to reduce the cost and simplify the fabrication for the fiber scanner without compromising performance or stability (<0.3 µm drift over 84 hours). A custom long-wavelength (∼1.08 µm) femtosecond fiber laser is coupled into several meters of fiber to realize a flexible, hand-held device for long-wavelength multiphoton microendoscopy.

Topographic Measurement of the Subretinal Pigment Epithelium Space in Normal Aging and Age-Related Macular Degeneration Using High-Resolution OCT.

A micrometer scale hyporeflective band within the retinal pigment epithelium basal lamina - Bruch's membrane complex (RPE-BL-BrM) was topographically measured in aging and age-related macular degeneration (AMD). In a prospective cross-sectional study, 90 normal eyes from 76 subjects (range = 23-90 years) and 53 dry AMD eyes from 47 subjects (range = 62-91 years) were enrolled. Isotropic volume raster scans over 6mm × 6mm (500 × 500 A-scans) were acquired using a high-resolution (2.7 µm axial resolution) spectral-domain optical coherence tomography (SD-OCT) prototype instrument. Six consecutive optical coherence tomography (OCT) volumes were computationally motion-corrected and fused to improve feature visibility. A boundary regression neural network was developed to measure hyporeflective band thickness. Topographic dependence was evaluated over a 6-mm-diameter Early Treatment Diabetic Retinopathy Study (ETDRS) grid. The hyporeflective band thickness map (median of 4.3 µm and 7.8 µm in normal and AMD eyes, respectively) is thicker below and radially symmetric around the fovea. In normal eyes, age-associated differences occur within 0.7 to 2.3mm from the foveal center (P < 0.05). In AMD eyes, the hyporeflective band is hypothesized to be basal laminar deposits (BLamDs) and is thicker within the 3-mm ETDRS circle (P < 0.0002) compared with normal eyes. The inner ring is the most sensitive location to detect age versus AMD-associated changes within the RPE-BL-BrM. AMD eyes with subretinal drusenoid deposits (SDDs) have a significantly thicker hyporeflective band (P < 0.001) than those without SDDs. The hyporeflective band is a quantifiable biomarker which differentiates AMD from aging. Longitudinal studies are warranted. The hyporeflective band may be a useful biomarker for risk stratification and disease progression.

Miniaturized Head-Mount Doppler Optical Coherence Tomography Scope for Freely Moving Mouse.

This study presents a miniaturized head-mount optical coherence tomography (OCT) system tailored for high-resolution brain imaging in freely moving mice, providing an advanced noninvasive imaging tool in neuroscience research. Leveraging optical coherence tomography technology, the system enables depth-resolved imaging and integrates functional OCT extensions, including angiography and Doppler imaging. Remarkably lightweight at 1.5 g, the device allows for the preservation of natural mouse behavior during imaging sessions. With a maximum 4 × 4 mm field of view and 7.4 μm axial resolution, the system offers reliable imaging capabilities. Noteworthy features include focal adjustability, rotary joint integration for fiber-twist-free operation, and a high-speed swept-source OCT laser at 200 kHz, facilitating real-time imaging. By providing insights into brain mechanisms and neurological disorders without disrupting natural behavior, this innovative system holds promise as a powerful tool in neuroscience research. Its compact design and comprehensive imaging capabilities make it well-suited for studying various brain regions and dynamic processes, contributing significantly to our understanding of brain function and pathology.

Axially swept open-top light-sheet microscopy for densely labeled clinical specimens.

Open-top light-sheet (OTLS) microscopy offers rapid 3D imaging of large optically cleared specimens. This enables nondestructive 3D pathology, which provides key advantages over conventional slide-based histology including comprehensive sampling without tissue sectioning/destruction and visualization of diagnostically important 3D structures. With 3D pathology, clinical specimens are often labeled with small-molecule stains that broadly target nucleic acids and proteins, mimicking conventional hematoxylin and eosin (H&E) dyes. Tight optical sectioning helps to minimize out-of-focus fluorescence for high-contrast imaging in these densely labeled tissues but has been challenging to achieve in OTLS systems due to trade-offs between optical sectioning and field of view. Here we present an OTLS microscope with voice-coil-based axial sweeping to circumvent this trade-off, achieving 2 µm axial resolution over a 750 × 375 µm field of view. We implement our design in a non-orthogonal dual-objective (NODO) architecture, which enables a 10-mm working distance with minimal sensitivity to refractive index mismatches, for high-contrast 3D imaging of clinical specimens.

Smartphone-based optical sectioning (SOS) microscopy with a telecentric design for fluorescence imaging.

We propose a smartphone-based optical sectioning (SOS) microscope based on the HiLo technique, with a single smartphone replacing a high-cost illumination source and a camera sensor. We built our SOS with off-the-shelf optical, mechanical cage systems with 3D-printed adapters to seamlessly integrate the smartphone with the SOS main body. The liquid light guide can be integrated with the adapter, guiding the smartphone's LED light to the digital mirror device (DMD) with neglectable loss. We used an electrically tuneable lens (ETL) instead of a mechanical translation stage to realise low-cost axial scanning. The ETL was conjugated to the objective lens's back pupil plane (BPP) to construct a telecentric design by a 4f configuration to maintain the lateral magnification for different axial positions. SOS has a 571.5µm telecentric scanning range and an 11.7µm axial resolution. The broadband smartphone LED torch can effectively excite fluorescent polystyrene (PS) beads. We successfully used SOS for high-contrast fluorescent PS beads imaging with different wavelengths and optical sectioning imaging of multilayer fluorescent PS beads. To our knowledge, the proposed SOS is the first smartphone-based HiLo optical sectioning microscopy (£1965), which can save around £7035 compared with a traditional HiLo system (£9000). It is a powerful tool for biomedical research in resource-limited areas.

Wearable optical coherence tomography angiography probe for freely moving mice.

Optical coherence tomography (OCT) is an emerging optical imaging technology that holds great potential in medical and biological applications. Apart from its conventional ophthalmic uses, it has found extensive applications in studying various brain activities and disorders in anesthetized/restricted rodents, with a particular focus on visualizing brain blood vessel morphology and function. However, developing a compact wearable OCT probe for studying the brain activity/disorders in freely moving rodents is challenging due to the requirements for stability and lightweight design. Here, we report a robust wearable OCT probe, which, to the best of our knowledge, is the first wearable OCT angiography probe capable of long-term monitoring of mouse brain blood flow. This wearable imaging probe has a maximum scanning speed of 76 kHz, with a 12 µm axial resolution, 5.5 µm lateral resolution, and a large field of view (FOV) of 4 mm × 4 mm. It offers easy assembly and stable imaging, enabling it to capture brain vessels in freely moving rodents. We tested this probe to monitor cerebral hemodynamics for up to 4 hours during the acute ischemic phase after photothrombotic stroke in mice, highlighting the reliability and long-term stability of our probe. This work contributes to the advancement of wearable biomedical imaging.

High-axial-resolution speckle-free holographic reconstruction via cylindrical quadratic phase method and temporal focusing.

Holographic techniques enable precise laser manipulation, but suffer from two considerable limitations: speckle and deterioration of axial distribution. Here, we propose a cylindrical quadratic phase (CQP) method with temporal focusing (TF) to generate speckle-free holographic illumination with high axial resolution. TF-CQP utilizes a superposed cylindrical phase as the initial guess to iteratively optimize phase hologram, realizing speckle-free holographic reconstruction on the target focal plane and eliminating secondary focus on the defocused planes. TF-CQP further disperses defocused beams symmetrically by a blazed grating, placed conjugate to the focal plane, which enhances axial confinement. Simulation and experimental results show that TF-CQP reconstructs speckle-free illumination with arbitrary shapes and <10 µm axial resolution. Compared to TF-GS (Gerchberg-Saxton algorithm), widely used in holographic optogenetics, TF-CQP shows increased uniformity of 200% and improved modulation efficiency of 32.33% for parallel holographic illumination, as well as a 10% increment in axial resolution.

Robotic-OCT guided inspection and microsurgery of monolithic storage devices

Data recovery from monolithic storage devices (MSDs) is in high demand for legal or business purposes. However, the conventional data recovery methods are destructive, complicated, and time-consuming. We develop a robotic-arm-assisted optical coherence tomography (robotic-OCT) for non-destructive inspection of MSDs, offering ~7 μm lateral resolution, ~4 μm axial resolution and an adjustable field-of-view to accommodate various MSD sizes. Using a continuous scanning strategy, robotic-OCT achieves automated volumetric imaging of a micro-SD card in ~37 seconds, significantly faster than the traditional stop-and-stare scanning that typically takes tens of minutes. We also demonstrate the robotic-OCT-guided laser ablation as a microsurgical tool for targeted area removal with precision of ±10 μm and accuracy of ~50 μm, eliminating the need to remove the entire insulating layer and operator intervention, thus greatly improving the data recovery efficiency. This work has diverse potential applications in digital forensics, failure analysis, materials testing, and quality control.

Mapping nanoscale topographic features in thick tissues with speckle diffraction tomography

Resolving three-dimensional morphological features in thick specimens remains a significant challenge for label-free imaging. We report a new speckle diffraction tomography (SDT) approach that can image thick biological specimens with ~500 nm lateral resolution and ~1 μm axial resolution in a reflection geometry. In SDT, multiple-scattering background is rejected through spatiotemporal gating provided by dynamic speckle-field interferometry, while depth-resolved refractive index maps are reconstructed by developing a comprehensive inverse-scattering model that also considers specimen-induced aberrations. Benefiting from the high-resolution and full-field quantitative imaging capabilities of SDT, we successfully imaged red blood cells and quantified their membrane fluctuations behind a turbid medium with a thickness of 2.8 scattering mean-free paths. Most importantly, we performed volumetric imaging of cornea inside an ex vivo rat eye and quantified its optical properties, including the mapping of nanoscale topographic features of Dua’s and Descemet’s membranes that had not been previously visualized.

Miniature side-view dual axes confocal endomicroscope for repetitive in vivo imaging.

A side-view dual axes confocal endomicroscope is demonstrated that can be inserted repetitively in hollow organs of genetically engineered mice for in vivo real-time imaging in horizontal and vertical planes. Near infrared (NIR) excitation at λex = 785 nm was used. A monolithic 3-axis parametric resonance scan mirror was fabricated using micro-electro-mechanical systems (MEMS) technology to perform post-objective scanning in the distal end of a 4.19 mm diameter instrument. Torsional and serpentine springs were designed to "switch" the mode of imaging between vertical and horizontal planes by tuning the actuation frequency. This system demonstrated real-time in-vivo images in horizontal and vertical planes with 310 µm depth and 1.75 and 7.5 µm lateral and axial resolution. Individual cells and discrete mucosal structures could be identified.

Reliability of Retinal Layer Annotation with a Novel, High-Resolution Optical Coherence Tomography Device: A Comparative Study.

Optical coherence tomography (OCT) enables in vivo diagnostics of individual retinal layers in the living human eye. However, improved imaging resolution could aid diagnosis and monitoring of retinal diseases and identify potential new imaging biomarkers. The investigational high-resolution OCT platform (High-Res OCT; 853 nm central wavelength, 3 µm axial-resolution) has an improved axial resolution by shifting the central wavelength and increasing the light source bandwidth compared to a conventional OCT device (880 nm central wavelength, 7 µm axial-resolution). To assess the possible benefit of a higher resolution, we compared the retest reliability of retinal layer annotation from conventional and High-Res OCT, evaluated the use of High-Res OCT in patients with age-related macular degeneration (AMD), and assessed differences of both devices on subjective image quality. Thirty eyes of 30 patients with early/intermediate AMD (iAMD; mean age 75 ± 8 years) and 30 eyes of 30 age-similar subjects without macular changes (62 ± 17 years) underwent identical OCT imaging on both devices. Inter- and intra-reader reliability were analyzed for manual retinal layer annotation using EyeLab. Central OCT B-scans were graded for image quality by two graders and a mean-opinion-score (MOS) was formed and evaluated. Inter- and intra-reader reliability were higher for High-Res OCT (greatest benefit for inter-reader reliability: ganglion cell layer; for intra-reader reliability: retinal nerve fiber layer). High-Res OCT was significantly associated with an improved MOS (MOS 9/8, Z-value = 5.4, p < 0.01) mainly due to improved subjective resolution (9/7, Z-Value 6.2, p < 0.01). The retinal pigment epithelium drusen complex showed a trend towards improved retest reliability in High-Res OCT in iAMD eyes but without statistical significance. Improved axial resolution of the High-Res OCT benefits retest reliability of retinal layer annotation and improves perceived image quality and resolution. Automated image analysis algorithms could also benefit from the increased image resolution.

High-resolution and high-speed 3D tracking of microrobots using a fluorescent light field microscope.

Imaging and tracking are crucial for microrobots which navigate through complex 3D environments. Fluorescent imaging (FI) by microscope offers a high-resolution and high-sensitive imaging method to study the property of microrobots. However, conventional microscope suffers from shallow depth of field (DOF) and lacks 3D imaging capability. We proposed a high-resolution and high-speed 3D tracking method for microrobots based on a fluorescent light field microscope (FLFM). We designed the FLFM system according to the size of a representative helical microrobot (150 μm body length, 50 μm screw diameter), and studied the system's performance. We also proposed a 3D tracking algorithm for microrobots using digital refocusing. We validated the method by simulations and built an FLFM system to perform the tracking experiments of microrobots with representative size. Our 3D tracking method achieves a 30 fps data acquisition rate, 10 μm lateral resolution and approximately 40 μm axial resolution over a volume of 1,200×1,200×326 μm3. Results indicate that the accuracy of the method can reach about 9 μm. Compared with the FI by a conventional microscope, the FLFM-based method gains wider DOF and 3D imaging capability with a single-shot image. The tracking method succeeds in providing the trajectory of the microrobot with a good lateral resolution.

Comparison of Fabrication Methods for Fiber‐Optic Ultrasound Transmitters Using Candle‐Soot Nanoparticles

AbstractCandle‐soot nanoparticles (CSNPs) have shown great promise for fabricating optical ultrasound (OpUS) transmitters. They have a facile, inexpensive synthesis whilst their unique, porous structure enables a fast heat diffusion rate which aids high‐frequency ultrasound generation necessary for high‐resolution clinical imaging. These composites have demonstrated high ultrasound generation performance showing clinically relevant detail, when applied as macroscale OpUS transmitters comprising both concave and planar surfaces, however, less research has been invested into the translation of this material's technology to fabricate fiber‐optic transmitters for image guidance of minimally invasive interventions. Here, are reported two fabrication methods of nanocomposites composed of CSNPs embedded within polydimethylsiloxane (PDMS) deposited onto fiber‐optic end‐faces using two different optimized fabrication methods: “All‐in‐One” and “Direct Deposition.” Both types of nanocomposite exhibit a smooth, black domed structure with a maximum dome thickness of 50 µm, broadband optical absorption (&gt;98% between 500 and 1400 nm) and both nanocomposites generated high peak‐to‐peak ultrasound pressures (&gt;3 MPa) and wide bandwidths (&gt;29 MHz). Further, high‐resolution (&lt;40 µm axial resolution) B‐mode ultrasound imaging of ex vivo lamb brain tissue demonstrating how CSNP‐PDMS OpUS transmitters can allow for high fidelity minimally invasive imaging of biological tissues is demonstrated.

Adaptive optics scanning laser ophthalmoscopy and optical coherence tomography (AO-SLO-OCT) system for in vivo mouse retina imaging.

Optical coherence tomography (OCT) and scanning laser ophthalmoscopy (SLO) are imaging technologies invented in the 1980s that have revolutionized the field of in vivo retinal diagnostics and are now commonly used in ophthalmology clinics as well as in vision science research. Adaptive optics (AO) technology enables high-fidelity correction of ocular aberrations, resulting in improved resolution and sensitivity for both SLO and OCT systems. The potential of gathering multi-modal cellular-resolution information in a single instrument is of great interest to the ophthalmic imaging community. Although similar instruments have been developed for imaging the human retina, developing such a system for mice will benefit basic science research and should help with further dissemination of AO technology. Here, we present our work integrating OCT into an existing mouse retinal AO-SLO system, resulting in a multi-modal AO-enhanced imaging system of the living mouse eye. The new system allows either independent or simultaneous data acquisition of AO-SLO and AO-OCT, depending on the requirements of specific scientific experiments. The system allows a data acquisition speed of 200 kHz A-scans/pixel rate for OCT and SLO, respectively. It offers ∼6 µm axial resolution for AO-OCT and a ∼1 µm lateral resolution for AO-SLO-OCT imaging.

40 MHz Swept-Source Optical Coherence Tomography at 1060 nm Using a Time-Stretch and Supercontinuum Spectral Broadening Dynamics

We present an akinetically swept light source based on a spectrally broadened mode-locked laser in all normal dispersion optical fiber followed by a linear dispersive time stretch and a regenerative amplification stage. Developed for ultrafast swept-source optical coherence tomography (SS-OCT), the source enables the demonstration of A-scan rate at 40 MHz with a 12 μm axial resolution. Through A-scans captured from a fast-moving object, we demonstrate that high-speed OCT is possible at 1 μm wavelength in an architecture which is not intrinsically bandwidth limited. The source therefore shows a promising route to achieving high axial resolution in high-speed swept-source OCT.

Classifying breast cancer in ultrahigh-resolution optical coherence tomography images using convolutional neural networks.

Optical coherence tomography (OCT) is being investigated in breast cancer diagnostics as a real-time histology evaluation tool. We present a customized deep convolutional neural network (CNN) for classification of breast tissues in OCT B-scans. Images of human breast samples from mastectomies and breast reductions were acquired using a custom ultrahigh-resolution OCT system with 2.72 µm axial resolution and 5.52 µm lateral resolution. The network achieved 96.7% accuracy, 92% sensitivity, and 99.7% specificity on a dataset of 23 patients. The usage of deep learning will be important for the practical integration of OCT into clinical practice.

Flexible transparent CMUT arrays for photoacoustic tomography.

This paper reports the fabrication and characterization of the first flexible transparent capacitive micromachined ultrasound transducer (CMUT) array for through-illumination photoacoustic tomography. Fabricated based on an adhesive wafer bonding technique and a PDMS backfill approach, the array has a maximum transparency of 67% in visible light range and can be bent to a radius of curvature of less than 5 mm without the structural layers being damaged. With a center frequency of 3.5 MHz, 80% fractional bandwidth, and noise equivalent pressure (NEP) of 62 mPa/H z, the array was successfully used in limited-view photoacoustic tomography of a 100 µm wire target, demonstrating lateral and axial resolutions of 293 µm and 382 µm, respectively, with 46 dB signal-to-noise ratio. Additionally, deep tissue photoacoustic tomography was also demonstrated on a blood tube within a chicken tissue using the fabricated CMUT arrays.

High speed, long range, deep penetration swept source OCT for structural and angiographic imaging of the anterior eye

This study reports the development of prototype swept-source optical coherence tomography (SS-OCT) technology for imaging the anterior eye. Advances in vertical-cavity surface-emitting laser (VCSEL) light sources, signal processing, optics and mechanical designs, enable a unique combination of high speed, long range, and deep penetration that addresses the challenges of anterior eye imaging. We demonstrate SS-OCT with a 325 kHz A-scan rate, 12.2 µm axial resolution (in air), and 15.5 mm depth range (in air) at 1310 nm wavelength. The ultrahigh 325 kHz A-scan rate not only facilitates biometry measurements by minimizing acquisition time and thus reducing motion, but also enables volumetric OCT for comprehensive structural analysis and OCT angiography (OCTA) for visualizing vasculature. The 15.5 mm (~ 11.6 mm in tissue) depth range spans all optical surfaces from the anterior cornea to the posterior lens capsule. The 1310 nm wavelength range enables structural OCT and OCTA deep in the sclera and through the iris. Achieving high speed and long range requires linearizing the VCSEL wavenumber sweep to efficiently utilize analog-to-digital conversion bandwidth. Dual channel recording of the OCT and calibration interferometer fringe signals, as well as sweep to sweep wavenumber compensation, is used to achieve invariant 12.2 µm (~ 9.1 µm in tissue) axial resolution and optimum point spread function throughout the depth range. Dynamic focusing using a tunable liquid lens extends the effective depth of field while preserving the lateral resolution. Improved optical and mechanical design, including parallax “split view” iris cameras and stable, ergonomic patient interface, facilitates accurate instrument positioning, reduces patient motion, and leads to improved imaging data yield and measurement accuracy. We present structural and angiographic OCT images of the anterior eye, demonstrating the unique imaging capabilities using representative scanning protocols which may be relevant to future research and clinical applications.

Airy light-sheet Raman imaging.

Light-sheet fluorescence microscopy has greatly improved the speed and overall photostability of optically sectioning cellular and multi-cellular specimens. Similar gains have also been conferred by light-sheet Raman imaging; these schemes, however, rely on diffraction limited Gaussian beams that hinder the uniformity and size of the imaging field-of-view, and, as such, the resulting throughput rates. Here, we demonstrate that a digitally scanned Airy beam increases the Raman imaging throughput rates by more than an order of magnitude than conventional diffraction-limited beams. Overall, this, spectrometer-less, approach enabled 3D imaging of microparticles with high contrast and 1 µm axial resolution at 300 msec integration times per plane and orders of magnitude lower irradiation density than coherent Raman imaging schemes. We detail the apparatus and its performance, as well as its compatibility with fluorescence light-sheet and quantitative-phase imaging towards rapid and low phototoxicity multimodal imaging.

INTERMEDIATE AND DEEP CAPILLARY PLEXUSES IN MACHINE LEARNING SEGMENTATION OF HIGH-RESOLUTION OPTICAL COHERENCE TOMOGRAPHY IMAGING.

To describe imaging produced by machine learning-based segmentation of high-resolution optical coherence tomography imaging of the intermediate capillary plexus and deep capillary plexus, layers of vessels not imaged well by dye-based angiography. Three healthy subjects with no ocular problems were imaged with spectral domain optical coherence tomography using an instrument with a scanning speed of 85,000 A-scans per second and 3 µm axial optical resolution. A random forest segmentation strategy was used to segment the intermediate capillary plexus and deep capillary plexus. The depth-resolved imaging data was visualized with the help of volume rendering. The high-resolution optical coherence tomography showed the intermediate capillary plexus and deep capillary plexus at the outer borders of the inner nuclear layer. These vessels could be visualized with unprecedented detail in three dimensions. There were multiple bridging vessels connecting to the whorl-like patterns of capillary mesh of the deep capillary plexus, a feature only previously imaged in histologic evaluation of excised eyes. High-resolution optical coherence tomography, machine learning, and advanced image display techniques have wide relevancy in studying the retina in health and disease. Application of this approach has provided images of the deeper vascular layers of the eye that approximate histologic imaging, but noninvasively.