On-demand expansion fluorescence and photoacoustic microscopy (ExFLPAM).

Expansion microscopy (ExM) is a promising technology that enables nanoscale imaging on conventional optical microscopes by physically magnifying the specimens. Here, we report the development of a strategy that enables i) on-demand labeling of subcellular organelles in live cells for ExM through transfection of fluorescent proteins that are well-retained during the expansion procedure; and ii) non-fluorescent chromogenic color-development towards efficient bright-field and photoacoustic imaging in both planar and volumetric formats, which is applicable to both cultured cells and biological tissues. Compared to the conventional ExM methods, our strategy provides an expanded toolkit, which we term as expansion fluorescence and photoacoustic microscopy (ExFLPAM), by allowing on-demand fluorescent protein labeling of cultured cells, as well as non-fluorescent absorption contrast-imaging of biological samples.

Longitudinal intravital imaging of mouse placenta.

Studying placental functions is crucial for understanding pregnancy complications. However, imaging placenta is challenging due to its depth, volume, and motion distortions. In this study, we have developed an implantable placenta window in mice that enables high-resolution photoacoustic and fluorescence imaging of placental development throughout the pregnancy. The placenta window exhibits excellent transparency for light and sound. By combining the placenta window with ultrafast functional photoacoustic microscopy, we were able to investigate the placental development during the entire mouse pregnancy, providing unprecedented spatiotemporal details. Consequently, we examined the acute responses of the placenta to alcohol consumption and cardiac arrest, as well as chronic abnormalities in an inflammation model. We have also observed viral gene delivery at the single-cell level and chemical diffusion through the placenta by using fluorescence imaging. Our results demonstrate that intravital imaging through the placenta window can be a powerful tool for studying placenta functions and understanding the placental origins of adverse pregnancy outcomes.

Self-enhancing sono-inks enable deep-penetration acoustic volumetric printing.

Volumetric printing, an emerging additive manufacturing technique, builds objects with enhanced printing speed and surface quality by forgoing the stepwise ink-renewal step. Existing volumetric printing techniques almost exclusively rely on light energy to trigger photopolymerization in transparent inks, limiting material choices and build sizes. We report a self-enhancing sonicated ink (or sono-ink) design and corresponding focused-ultrasound writing technique for deep-penetration acoustic volumetric printing (DAVP). We used experiments and acoustic modeling to study the frequency and scanning rate-dependent acoustic printing behaviors. DAVP achieves the key features of low acoustic streaming, rapid sonothermal polymerization, and large printing depth, enabling the printing of volumetric hydrogels and nanocomposites with various shapes regardless of their optical properties. DAVP also allows printing at centimeter depths through biological tissues, paving the way toward minimally invasive medicine.

Theoretical and experimental study on the detection limit of the micro-ring resonator based ultrasound point detectors.

Combining the diffusive laser excitation and the photoacoustic signals detection, photoacoustic computed tomography (PACT) is uniquely suited for deep tissue imaging. A diffraction-limited ultrasound point detector is highly desirable for maximizing the spatial resolution and the field-of-view of the reconstructed volumetric images. Among all the available ultrasound detectors, micro-ring resonator (MRR) based ultrasound detectors offer the lowest area-normalized limit of detection (nLOD) in a miniature form-factor, making it an ideal candidate as an ultrasound point detector. However, despite their wide adoption for photoacoustic imaging, the underlying signal transduction process has not been systematically studied yet. Here we report a comprehensive theoretical model capturing the transduction of incident acoustic signals into digital data, and the associated noise propagation process, using experimentally calibrated key process parameters. The theoretical model quantifies the signal-to-noise ratio (SNR) and the nLOD under the influence of the key process variables, including the quality factor (Q-factor) of the MRR and the driving wavelength. While asserting the need for higher Q-factors, the theoretical model further quantifies the optimal driving wavelength for optimizing the nLOD. Given the MRR with a Q-factor of 1 × 105, the theoretical model predicts an optimal SNR of 30.1 dB and a corresponding nLOD of 3.75 × 10-2 mPa mm2/Hz1/2, which are in good agreement with the experimental measurements of 31.0 dB and 3.39 × 10-2 mPa mm2/Hz1/2, respectively. The reported theoretical model can be used in guiding the optimization of MRR-based ultrasonic detectors and PA experimental conditions, in attaining higher imaging resolution and contrast. The optimized operating condition has been further validated by performing PACT imaging of a human hair phantom.

On-Demand Expansion Fluorescence and Photoacoustic Microscopy (ExFLPAM).

Expansion microscopy (ExM) is a promising technology that enables nanoscale imaging on conventional optical microscopes by physically magnifying the specimens. Here, we report the development of a strategy that enables i) on-demand labeling of subcellular organelles in live cells for ExM through transfection of fluorescent proteins that are well-retained during the expansion procedure; and ii) non-fluorescent chromogenic color-development towards efficient bright-field and photoacoustic imaging in both planar and volumetric formats, which is applicable to both cultured cells and biological tissues. Compared to the conventional ExM methods, our strategy provides an expanded toolkit, which we term as expansion fluorescence and photoacoustic microscopy (ExFLPAM), by allowing on-demand fluorescent protein labeling of cultured cells, as well as non-fluorescent absorption contrast-imaging of biological samples.

Whole-brain imaging of freely-moving zebrafish.

One of the holy grails of neuroscience is to record the activity of every neuron in the brain while an animal moves freely and performs complex behavioral tasks. While important steps forward have been taken recently in large-scale neural recording in rodent models, single neuron resolution across the entire mammalian brain remains elusive. In contrast the larval zebrafish offers great promise in this regard. Zebrafish are a vertebrate model with substantial homology to the mammalian brain, but their transparency allows whole-brain recordings of genetically-encoded fluorescent indicators at single-neuron resolution using optical microscopy techniques. Furthermore zebrafish begin to show a complex repertoire of natural behavior from an early age, including hunting small, fast-moving prey using visual cues. Until recently work to address the neural bases of these behaviors mostly relied on assays where the fish was immobilized under the microscope objective, and stimuli such as prey were presented virtually. However significant progress has recently been made in developing brain imaging techniques for zebrafish which are not immobilized. Here we discuss recent advances, focusing particularly on techniques based on light-field microscopy. We also draw attention to several important outstanding issues which remain to be addressed to increase the ecological validity of the results obtained.

Real-time whole-brain imaging of hemodynamics and oxygenation at micro-vessel resolution with ultrafast wide-field photoacoustic microscopy.

High-speed high-resolution imaging of the whole-brain hemodynamics is critically important to facilitating neurovascular research. High imaging speed and image quality are crucial to visualizing real-time hemodynamics in complex brain vascular networks, and tracking fast pathophysiological activities at the microvessel level, which will enable advances in current queries in neurovascular and brain metabolism research, including stroke, dementia, and acute brain injury. Further, real-time imaging of oxygen saturation of hemoglobin (sO2) can capture fast-paced oxygen delivery dynamics, which is needed to solve pertinent questions in these fields and beyond. Here, we present a novel ultrafast functional photoacoustic microscopy (UFF-PAM) to image the whole-brain hemodynamics and oxygenation. UFF-PAM takes advantage of several key engineering innovations, including stimulated Raman scattering (SRS) based dual-wavelength laser excitation, water-immersible 12-facet-polygon scanner, high-sensitivity ultrasound transducer, and deep-learning-based image upsampling. A volumetric imaging rate of 2 Hz has been achieved over a field of view (FOV) of 11 × 7.5 × 1.5 mm3 with a high spatial resolution of ~10 µm. Using the UFF-PAM system, we have demonstrated proof-of-concept studies on the mouse brains in response to systemic hypoxia, sodium nitroprusside, and stroke. We observed the mouse brain's fast morphological and functional changes over the entire cortex, including vasoconstriction, vasodilation, and deoxygenation. More interestingly, for the first time, with the whole-brain FOV and micro-vessel resolution, we captured the vasoconstriction and hypoxia simultaneously in the spreading depolarization (SD) wave. We expect the new imaging technology will provide a great potential for fundamental brain research under various pathological and physiological conditions.

High-Frequency 3D Photoacoustic Computed Tomography Using an Optical Microring Resonator.

3D photoacoustic computed tomography (3D-PACT) has made great advances in volumetric imaging of biological tissues, with high spatial-temporal resolutions and large penetration depth. The development of 3D-PACT requires high-performance acoustic sensors with a small size, large detection bandwidth, and high sensitivity. In this work, we present a new high-frequency 3D-PACT system that uses a micro-ring resonator (MRR) as the acoustic sensor. The MRR sensor has a size of 80 µm in diameter, and was fabricated using the nanoimprint lithography technology. Using the MRR sensor, we have developed a transmission-mode 3D-PACT system that has achieved a detection bandwidth of ~23 MHz, an imaging depth of ~8 mm, a lateral resolution of 114 µm, and an axial resolution of 57 µm. We have demonstrated the 3D PACT's performance on in vitro phantoms, ex vivo mouse brain, and in vivo mouse ear and tadpole. The MRR-based 3D-PACT system can be a promising tool for structural, functional, and molecular imaging of biological tissues at depths.

High-speed functional photoacoustic microscopy using a water-immersible two-axis torsion-bending scanner.

Optical-resolution photoacoustic microscopy (OR-PAM) can provide functional, anatomical, and molecular images at micrometer level resolution with an imaging depth of less than 1 mm in tissue. However, the imaging speed of traditional OR-PAM is often low due to the point-by-point mechanical scanning and cannot capture time-sensitive dynamic information. In this work, we demonstrate a recent effort in improving the imaging speed of OR-PAM, using a newly developed water-immersible two-axis scanner. Driven by water-compatible electromagnetic actuation force, the new scanning mirror employs a novel torsion-bending mechanism to achieve fast 2D scanning. The torsion scanning along the fast-axis works in the resonant model, and the bending scanning along the slow-axis operate at the quasi-static mode. The scanning speed and scanning range along the two axes can be independently adjusted. Steered by the two-axis torsion-bending scanning mirror immersed in water, the focused excitation light and the generated acoustic wave can be confocally aligned over the entire imaging area. Thus, a high imaging speed can be achieved without sacrificing the detection sensitivity. Equipped with the torsion-bending scanner, the high-speed OR-PAM system has achieved a cross-sectional frame rate of 400 Hz, and a volumetric imaging speed of 1 Hz over a field of view of 1.5 × 2.5 mm2. We have also demonstrated high-speed OR-PAM of the hemodynamic changes in response to pharmaceutical and physiological challenges in small animal models in vivo. We expect the torsion-bending scanner based OR-PAM will find matched biomedical studies of tissue dynamics.

An Ultra-Long-Life Flexible Lithium-Sulfur Battery with Lithium Cloth Anode and Polysulfone-Functionalized Separator.

Flexible and high-performance batteries are urgently required for powering flexible/wearable electronics. Lithium-sulfur batteries with a very high energy density are a promising candidate for high-energy-density flexible power source. Here, we report flexible lithium-sulfur full cells consisting of ultrastable lithium cloth anodes, polysulfone-functionalized separators, and free-standing sulfur/graphene/boron nitride nanosheet cathodes. The carbon cloth decorated with lithiophilic three-dimensional MnO2 nanosheets not only provides the lithium anodes with an excellent flexibility but also limits the growth of the lithium dendrites during cycling, as revealed by theoretical calculations. Commercial separators are functionalized with polysulfone (PSU) via a phase inversion strategy, resulting in an improved thermal stability and smaller pore size. Due to the synergistic effect of the PSU-functionalized separators and boron nitride-graphene interlayers, the shuttle of the polysulfides is significantly inhibited. Because of successful control of the shuttle effect and dendrite formation, the flexible lithium-sulfur full cells exhibit excellent mechanical flexibility and outstanding electrochemical performance, which shows a superlong lifetime of 800 cycles in the folded state and a high areal capacity of 5.13 mAh cm-2. We envision that the flexible strategy presented herein holds promise as a versatile and scalable platform for large-scale development of high-performance flexible batteries.

Highly sensitive fiber-optic accelerometer using a micro suspended-core fiber.

A compact fiber-optic accelerometer was proposed and demonstrated experimentally based on Fabry-Perot interference (FPI). The device consists of a suspended-core fiber embedded in a hollow-core fiber, forming an enclosed cavity structure. A short section of multi-mode fiber (MMF) was spliced on the leading-in single-mode fiber (SMF), which worked as a micro lens to focus the light to decrease the transmission loss. A well-defined interference spectrum was achieved by a low-fitness FP interferometer formed by both the end-face of lead-in fiber and the end-face of suspended-core fiber. Thanks to the outstretched FP cavity by suspended-core fiber, the sensor is highly sensitive to vibration along the fiber axis. Moreover, a one-dimensional mechanical transducer was used to improve the frequency band of the sensor. By the side-band filtering technology, the vibration was detected and analyzed by a simple intensity interrogation technology.

Sensitivity-Improved Ultrasonic Sensor for 3D Imaging of Seismic Physical Model Using a Compact Microcavity.

A sensitivity-improved ultrasonic sensor is proposed and demonstrated experimentally in this present study. The device is comprised only a fiber-optic microcavity that is formed by discharging a short section of hollow core fiber (HCF). The key to ensuring the success of the sensor relies on the preprocessing of hydrogen loading for HCF. When discharging the HCF, the hydrogen is heated up during the formation of the air bubble, which enlarges the bubble diameter, smoothens its surfaces simultaneously and decreases Young's modulus of the material of the bubble. Ultimately, this results in the probe being highly sensitive to ultrasound with a SNR of 69.28 dB. Once the compact air cavity is formed between the end face of the leading-in fiber and the top wall of the bubble, a well-defined interference spectrum is achieved based on the Fabry⁻Perot interference. By using spectral side-band filtering technology, we detect the ultrasonic waves reflected by the seismic physical model (SMF) and then reconstruct its three-dimensional image.

A Submerged Optical Fiber Ultrasonic Sensor Using Matched Fiber Bragg Gratings.

A novel kind of fiber optic ultrasonic sensor based on matching fiber Bragg gratings (FBGs) is proposed and demonstrated. The sensors consist of a pair of matching FBGs fixed to a special bracket. The bracket plays a role in stretching and squeezing the FBGs, with the push⁻pull effect efficiently coupling the ultrasonic signal to the sensor, thus, improving the sensor’s sensitivity. Side-band filtering technology-based intensity interrogation was used to detect ultrasounds in water. With the synergic effect of the matching FBGs, the sensor performed with a high signal-to-noise ratio (56.9 dB at 300 KHz, 53 dB at 1 MHz and 31.8 dB at 5 MHz) and the observed ultrasonic sinusoidal signals were undistorted and distinguishable in the time domain.

All-fiber 3D vector displacement (bending) sensor based on an eccentric FBG.

We demonstrate a fiber-optic 3D vector displacement sensor based on the monitoring of Bragg reflection from an eccentric grating inscribed in a depressed-cladding fiber using the femtosecond laser side-illumination and phase-mask technique. The compact sensing probe consists of a short section of depressed cladding fiber (DCF) containing eccentrically positioned fiber Bragg gratings. The eccentric grating breaks the cylindrical symmetry of the fiber cross-section and further has bending orientation-dependence. The generated fundamental resonance is strongly sensitive to bending of the fiber, and the direction of the bending plane can be determined from its responses. When integrated with axis strain monitoring, the sensor achieves a 3D vector displacement measurement via simple geometric analysis.

High-spatial-resolution ultrasonic sensor using a micro suspended-core fiber.

A high-resolution fiber-optic ultrasonic sensor based on a suspended-core fiber was designed and experimentally demonstrated. The intrinsic Fabry-Perot interferometer consisting of a micro suspended-core from acid corrosion of a grapefruit fiber proved highly sensitive to a wide range of ultrasonic wave (UW) frequencies. A compact interrogation system using spectral sideband filtering was constructed for UW detection. The sensor exhibited significantly improved spatial resolution and detection sensitivity by etching the suspended-core diameter to few microns. Sensor fabrication involves only fiber splicing and corrosion, which provide a self-shielding cladding surrounding and protecting the core from collisions. This sensor is an excellent candidate for high-quality UW detection.

Ultrasonic imaging of seismic physical models using a fringe visibility enhanced fiber-optic Fabry-Perot interferometric sensor.

A fringe visibility enhanced fiber-optic Fabry-Perot interferometer based ultrasonic sensor is proposed and experimentally demonstrated for seismic physical model imaging. The sensor consists of a graded index multimode fiber collimator and a PTFE (polytetrafluoroethylene) diaphragm to form a Fabry-Perot interferometer. Owing to the increase of the sensor's spectral sideband slope and the smaller Young's modulus of the PTFE diaphragm, a high response to both continuous and pulsed ultrasound with a high SNR of 42.92 dB in 300 kHz is achieved when the spectral sideband filter technique is used to interrogate the sensor. The ultrasonic reconstructed images can clearly differentiate the shape of models with a high resolution.

Orientation-dependent fiber-optic displacement sensor using a fiber Bragg grating inscribed in a side-hole fiber.

We propose and experimentally demonstrate an orientation-dependent fiber-optic bending sensor. The sensing probe consists of a fiber Bragg grating inscribed in both the fiber core and the surrounding cladding of a section of a side-hole fiber. We utilized a side-illumination technique using a femtosecond laser to achieve the grating structure formation. The transmission intensities of both resonances are highly sensitive bending of the fiber, and the bending response shows orientation dependence. The surrounding temperature fluctuation causes a wavelength shift, but not an intensity variation. Therefore, the proposed sensor can be employed for simultaneous measurement of bending and temperature.

Temperature-independent hygrometry using micromachined photonic crystal fiber.

An in-fiber Mach-Zehnder interferometer (MZI) is proposed and experimentally demonstrated for relative humidity (RH) and temperature measurements. The MZI is formed by a grapefruit-shaped photonic crystal fiber (G-PCF) cascaded with a short section of multimode fiber that serves as a mode coupler. To enhance sensitivity to humidity, femtosecond laser micromachining was performed to remove a portion of cladding of the G-PCF to expose its core to the ambient medium. The output interference spectrum is fast Fourier transformed to produce a spatial frequency spectrum that describes the intensity composition of the cladding modes in the MZI. In our investigation, it was observed that the interference dip intensity has a sensitivity of -0.077 dB/% RH to the change of RH in the range of 25%-80% RH, whereas the dip wavelength has a temperature sensitivity of ∼3.3 pm/°C in the range of 25°C-70°C. In addition, the dip intensity was insensitive to temperature. These characteristics have provided convenience in eliminating temperature cross talk and achieving accurate humidity measurement.

High-sensitivity optical fiber relative humidity probe with temperature calibration ability.

A high-sensitivity optical fiber relative humidity (RH) sensing probe with the ability of temperature calibration is proposed and experimentally demonstrated. It consists of a simple Fabry-Perot interferometer constructed by coating a layer of thin polyimide (PI) film on the end face of single-mode fiber and an upstream fiber Bragg grating (FBG). PI is one of the organic polymer humidity-sensitive materials with good comprehensive properties. The cascaded FBG is used for temperature calibration and elimination of the temperature cross-sensitivity in the process of measuring RH. Experimental results show that this sensing probe can realize simultaneous measurement of temperature and RH. The RH response sensitivity reaches up to 986.25 pm/%RH. This sensing probe with the advantages of simple structure, compact size, high sensitivity, easy packaging, and dual-parameter measurement has an extensive application prospect.

Compact gas refractometer based on a tapered four-core fiber.

A compact in-line interferometer is proposed and experimentally demonstrated for gas refractive index (GRI) measurement. The sensor comprises a tapered four-core fiber (TFCF) sandwiched between two single-mode fibers (SMFs), forming an in-line SMF-TFCF-SMF structure. The fiber taper acts as a bridge between the external GRI variation and the multimode interference within the TFCF segment. A high sensitivity of 1280.94 dB/refractive index unit is obtained in GRI measurement around 1.0. Temperature change only shifts the interference wavelength, and the cross-sensitivity of temperature can be ignored by intensity demodulation. The proposed gas refractometer, with its improved performance, can be a good candidate for chemical sensing or bio-sensing.