Researchers have developed a metalens-based photoacoustic microscopy system capable of 1.2 mm axial coverage, a substantial increase over the shallow depth of focus limiting current in-vivo tissue imaging. The new system uses a silicon nitride nanostructured metalens to switch between two focal modes at visible wavelengths, requiring only 0.87 V and 1.03 V and responding in 250 ms without mechanical parts. Demonstrated through high-resolution imaging of neovascularization in rat eyes with chemically induced corneal burns, the compact system provides “building blocks for future high-resolution volumetric imaging with an improved signal-to-noise ratio.”
Electrically Switchable Dual-Focus Metalens for Extended Depth Imaging
Achieving 1.2 mm axial coverage represents an advance in vivo photoacoustic imaging, enhancing axial coverage while preserving high lateral resolution. This extended range allows for comprehensive visualization of structures within living tissue, a capability demonstrated through imaging of rat eyes. The system utilizes a silicon nitride nanostructured metalens, dynamically switching between two distinct focal points without relying on mechanical components for adjustment. This electrical modulation enables high-resolution three-dimensional imaging across a significantly broader area than previously possible with comparable methods.
The dual-focus metalens operates at a wavelength of 532 nanometers, chosen for its strong absorption by hemoglobin, maximizing the photoacoustic signal contrast. This design incorporates nanostructures specifically engineered to direct light into two separate focal lengths, responding differently to right- and left-handed circularly polarized light.
The metalens achieves this focal shift in just 250 ms, facilitating swift 3D scanning and real-time monitoring of dynamic biological processes. In vivo microvascular imaging of a chemically induced corneal burn in rat eyes showcased the system’s capabilities, successfully capturing neovascularization, the formation of new blood vessels, with high resolution throughout the entire depth of the eye. Researchers observed improved resolution at depths of approximately one millimeter, confirming the effective shift in focal point achieved by the metalens.
The close agreement between vessel diameters and choroidal neovascularization lesion areas measured in the two focal modes indicates minimal polarization-dependent artifacts impacting the accuracy of the meta-PAM measurements. “In particular, the resolution in the thread at a depth of about 1 mm deeper (from #2 to #4) is also improved, confirming that the focus shifted by the metalens,” the study reports.
The polarization-dependent dual-focus metalens is mounted on a ring-type ultrasound transducer, further streamlining the system and eliminating the need for complex alignment procedures. This integration contributes to the system’s efficiency and ease of use. The development of this electrically tunable meta-PAM system, capable of switching imaging modes between two diffraction-limited optical foci, provides a platform for detailed investigation of ophthalmic diseases and potentially other biological applications requiring deep-tissue visualization. This technology builds on advances in flat optics and dispersion-engineered metasurfaces, offering a pathway toward more sophisticated and efficient imaging technologies.
Photoacoustic Microscopy Limitations with Conventional Optical Focusing
Conventional optical focusing in photoacoustic microscopy presents inherent limitations in imaging depth, a challenge addressed by exploring non-diffracting beams like Bessel and needle beams. While these approaches aim to extend imaging range, they often introduce high side lobes that diminish the signal-to-noise ratio and disperse laser energy, ultimately reducing signal intensity. This trade-off between depth and image quality has long constrained the effective use of photoacoustic microscopy for in vivo studies.
Photoacoustic microscopy uses pulsed optical excitation and ultrasonic detection to visualize optical absorption contrast within living tissues, capitalizing on the relatively weak scattering of ultrasound to achieve high-resolution imaging beyond the approximately one millimeter optical diffusion limit. Optical-resolution photoacoustic microscopy, in particular, relies on tightly focused laser beams to attain lateral resolution at the optical diffraction limit, enabling detailed visualization of microvascular networks.
However, maintaining this resolution at greater depths remains a persistent obstacle, as the focal spot size increases with distance from the surface, blurring the image and reducing contrast. The acquisition and processing of 3D PA datasets utilize tools like the 3D PHOVIS software in MATLAB, but even with advanced processing, the initial data quality is fundamentally limited by the optical system’s ability to maintain focus.
Efforts to improve depth penetration have led to investigations of various optical designs, including those incorporating flat optics and metasurfaces. These technologies offer the potential to manipulate light at the nanoscale, enabling the creation of customized beam profiles and focal characteristics. Recent work in this area has focused on developing metasurfaces capable of generating complex optical fields, such as Bessel beams and arbitrary photoacoustic fields, with the goal of overcoming the limitations of traditional focusing methods.
One study detailed in Ultrasonics demonstrated super-resolution ultrasound localization microscopy for visualizing ocular blood flow, highlighting ongoing efforts to enhance imaging capabilities within the eye. Another, published in Biophotonics, explored miniature probes utilizing double gradient-index lenses for photoacoustic microscopy, aiming to improve signal collection and resolution. These advancements, while promising, often require complex fabrication processes and may still suffer from trade-offs between resolution, depth, and signal strength.
High-Resolution 3D Imaging Needs in Biological Research
Measured lateral resolutions reached 3.1 and 3.7 micrometers for the two focal states, aligning closely with theoretical predictions of 3.0 and 3.5 micrometers in water, demonstrating the metalens’ capacity for high-resolution imaging across a relevant biological medium. The system’s ability to image intersecting 30-micrometer-thick black nylon threads at five distinct heights confirms its axial focal shift capability, a critical feature for volumetric scanning. This precise control over focal depth allows for detailed visualization of structures within living tissue, overcoming limitations inherent in conventional optical methods.
The potential for integration with other imaging modalities further expands the utility of this metalens-based photoacoustic microscopy (meta-PAM) system. For example, combining it with optical coherence tomography (OCT) could provide complementary contrast; OCT captures structural details via optical scattering, while PAM visualizes vascular and functional structures through optical absorption.
This combined approach is particularly beneficial in fields like ophthalmology and dermatology, where both structural and functional information are important for diagnosis. Seamless integration with two-photon microscopy is also possible, enabling high-resolution structural and functional imaging at extended depths, according to the research. Numerical simulations, conducted using the finite element method (FEM) solver COMSOL Multiphysics 6.1, underpinned the design and optimization of the metalens. These simulations employed periodic boundary conditions in the x- and y-directions and perfect boundary conditions in the z-direction, ensuring accurate modeling of light propagation.
The compact and tunable optics of the meta-PAM system facilitate these integrations, creating a versatile platform for advanced biomedical imaging applications. With its multimodal adaptability, meta-PAM promises to improve diagnostic accuracy and expand understanding of complex biological systems.
Silicon Nitride Nanostructures for Metalens Fabrication
Silicon nitride nanostructures are central to achieving 1.2 mm axial coverage in a new photoacoustic microscopy system, extending imaging depth significantly beyond the limitations of conventional optical methods. The material’s properties allow for the creation of dual-focus metalenses capable of switching between focal lengths without mechanical components, a critical feature for dynamic, three-dimensional imaging within living tissue. Researchers optimized the geometry of these nanostructures, varying height, period, length, and width, to function as half-wave plates, maximizing efficiency for both focal modes.
The selection of silicon nitride was deliberate, stemming from its suitability for photoacoustic imaging which relies on high-power pulsed lasers and detection of photoacoustic signals generated by optical absorption. Achieving high efficiency in both focal modes, designated F1 and F2, required careful design to operate optimally underwater, minimizing photoacoustic signal loss with a refractive index of 1.33.
Fabrication involved depositing a 700 or 1000 nanometer thick layer of silicon nitride onto a glass substrate using plasma enhanced chemical deposition. Measurements using ellipsometry and the Cauchy dispersion model confirmed the material’s refractive index, a key parameter in metalens design. Conversion efficiencies for both polarizations were intentionally designed to be identical. “We propose an electrically tunable, dual-focus metalens-based high-SNR PAM system capable of switching focal modes along the axial direction on a millisecond timescale, enabling high-resolution 3D imaging over a broad axial depth range,” the researchers state.
The system’s ability to switch focal modes is achieved with voltages of 0.87 V and 1.03 V, and a response time of 250 ms. The fabrication process, using established mass-production techniques, suggests a pathway toward widespread commercialization and integration of metasurfaces into practical optical technologies. The optimized design, with a period of 340 nanometers, represents a balance between efficiency and fabrication feasibility, ensuring reliable performance in aqueous environments.
Dual-Focus Metalens Design & Polarization Control
Achieving dynamic focal adjustment through an electrically tunable liquid crystal (LC) cell integrated with a metalens, the system eliminates the need for mechanical movement of optical components. This design allows for precise control of the incident polarization state, and therefore the focal length, by simply altering the electrical bias applied to the LC cell. Fabrication of the dual-focus metalens involves mounting and aligning it on an ultrasound transducer to detect photoacoustic waves generated by optical excitation.
Conversion efficiency at a 532 nanometer wavelength is maximized through optimized silicon nitride deposition, directly enhancing the metalens’ focusing capability, as detailed in Supplementary Figure S1. Independent modulation of light spin states, right circular polarization (RCP) and left circular polarization (LCP), relies on combining propagation and geometric (Pancharatnam-Berry) phase modulation techniques.
Researchers confirmed the half-wave plate characteristic of the chosen meta-atom by calculating the electric field under both x- and y-polarized incident light, with simulated electric field distributions and phase retardations detailed in Supplementary Figure S3. These simulations demonstrate the ability to achieve 2π phase modulation with varying meta-atom widths and lengths. The output polarization states passing through the LC when 45° rotated linearly polarized light is incident are also characterized.
To demonstrate dual-focus capability for both RCP and LCP light, two lens phase maps corresponding to focal lengths were designed. The Pancharatnam-Berry phase introduces equal and opposite phase retardations for LCP and RCP light, prompting rotation of the meta-atoms to match the phase maps for each polarization. Optical characterization of the electrically tunable dual-focus metalens involved integrating LC cells to modulate incident polarization states; the LC material, 4-cyano-4’pentylbiphenyl (5CB), alters its effective refractive index in response to an applied voltage.
Measurements reveal a response time of approximately 250 ms for this refractive index change, as shown in Supplementary Figure S4. A single-mode fiber and fiber collimator deliver the laser beam, while a polarizing beam splitter fixes the initial polarization state.
The metalens is coaxially assembled with a customized ring-type ultrasound transducer, and photoacoustic signals are amplified using a low-noise amplifier and collected by a data acquisition board and 12-bit digitizer operating at a 500 megasample per second rate. The system utilizes voltages of 0.87 V and 1.03 V to switch between focal modes, demonstrating low energy consumption for dynamic depth adjustment.
In Vivo Corneal Imaging Demonstrates 1.2mm Axial Coverage
The new metalens-based photoacoustic microscopy system achieves 1. This electrical modulation, achieved through a liquid crystal cell functioning as a quarter-wave plate, allows for focal mode switching on a millisecond timescale with a response time of 250 ms. Quantitative analysis of blood vessel diameters within the iris and corneal neovascularization regions confirmed the improved resolution; vessels in the CNV region measured approximately 18μm under the short-focus mode. Supplementary Figure S17, detailed in the supporting information, illustrates the method used for this quantification.
Beyond depth, the dual-focus capability addresses challenges posed by corneal neovascularization, a condition where new blood vessels grow into the cornea, creating height variations that complicate imaging. The system’s design enables the acquisition of high-resolution photoacoustic images across a broader field of view, as demonstrated by in vivo imaging of a normal rat eye using the short-focus mode, presented in the paper. This is a critical advantage for visualizing structures at varying depths within the complex corneal tissue.
The researchers also demonstrated the system’s performance by imaging nylon threads at different axial positions and sloped leaf skeletons, confirming enhanced resolution and contrast across a wider axial range. They write, highlighting the system’s speed and stability. The team’s work builds on previous advances in double gradient-index lenses photoacoustic microscopy, as noted in their citation of a 2018 Biophotonics paper.
The successful imaging of pathological neovascularization strictly confined to the cornea further validates the system’s effectiveness in visualizing disease processes. This capability has implications for understanding and potentially treating ophthalmic vascular diseases, offering a non-invasive method for detailed in vivo analysis. The system’s 250 ms response time allows for rapid 3D scanning, enabling real-time monitoring of dynamic processes within the eye.
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