An adaptive optics module for deep tissue multiphoton imaging in vivo.

Understanding complex biological systems requires visualizing structures and processes deep within living organisms. We developed a compact adaptive optics module and incorporated it into two- and three-photon fluorescence microscopes, to measure and correct tissue-induced aberrations. We resolved synaptic structures in deep cortical and subcortical areas of the mouse brain, and demonstrated high-resolution imaging of neuronal structures and somatosensory-evoked calcium responses in the mouse spinal cord at great depths in vivo.

Simultaneous multiplane imaging with reverberation two-photon microscopy.

Multiphoton microscopy has gained enormous popularity because of its unique capacity to provide high-resolution images from deep within scattering tissue. Here, we demonstrate video-rate multiplane imaging with two-photon microscopy by performing near-instantaneous axial scanning while maintaining three-dimensional micrometer-scale resolution. Our technique, termed reverberation microscopy, enables the monitoring of neuronal populations over large depth ranges and can be implemented as a simple add-on to a conventional design.

Polarization insensitive, metamaterial absorber-enhanced long-wave infrared detector.

Detecting low energy photons, such as photons in the long-wave infrared range, is a technically challenging proposition using naturally occurring materials. In order to address this challenge, we herein demonstrate a micro-bolometer featuring an integrated metamaterial absorber (MA), which takes advantage of the resonant absorption and frequency selective properties of the MA. Importantly, our micro-bolometer exhibits polarization insensitivity and high absorption due to a novel metal-insulator-metal (MIM) absorber design, operating at 8-12 µm wavelength. The metamaterial structures we report herein feature an interconnected design, optimized towards their application to micro-bolometer-based, long-wave infrared detection. The micro-bolometers were fabricated using a combination of conventional photolithography and electron beam lithography (EBL), the latter owing to the small feature sizes within the design. The absorption response was designed using the coupled mode theory (CMT) and the finite integration technique, with the fabricated devices characterized using Fourier-transform infrared spectroscopy (FTIR). The metamaterial-based micro-bolometer exhibits a responsivity of approximately 198 V/W over the 8-12 µm wavelength regime, detectivity of ∼ 0.6 × 109 Jones, thermal response time of ∼ 3.3 ms, and a noise equivalent temperature difference (NETD) of ∼33 mK under 1mA biasing current at room-temperature and atmosphere pressure. The ultimate detectivity and NETD are limited by Johnson noise and heat loss with thermal convection through air; however, further optimization could be achieved by reducing the thermal conductivity via vacuum packaging. Under vacuum conditions, the detectivity may be increased in excess of two-fold, to ∼ 1.5 × 109 Jones. Finally, an infrared image of a soldering iron was generated using a single-pixel imaging process, serving as proof-of-concept of this detection platform. The results presented in this work pave the road towards high-efficiency and frequency-selective detection in the long-wave infrared range through the integration of infrared MAs with micro-bolometers.

Adaptive optics in multiphoton microscopy: comparison of two, three and four photon fluorescence.

We demonstrate adaptive optics system based on nonlinear feedback from 3- and 4-photon fluorescence. The system is based on femtosecond pulses created by soliton self-frequency shift of a 1550-nm fiber-based femtosecond laser together with micro-electro-mechanical system (MEMS) phase spatial light modulator (SLM). We perturb the 1020-segment SLM using an orthogonal Walsh sequence basis set with a modified version of three-point phase shifting interferometry. We show the improvement after aberrations correction in 3-photon signal from fluorescent beads. In addition, we compare the improvement obtained in the same adaptive optical system for 2-, 3- and 4-photon fluorescence using dye pool. We show that signal improvement resulting from aberration correction grows exponentially as a function of the order of nonlinearity.

Field of view advantage of conjugate adaptive optics in microscopy applications.

The imaging performance of an optical microscope can be degraded by sample-induced aberrations. A general strategy to undo the effect of these aberrations is to apply wavefront correction with a deformable mirror (DM). In most cases the DM is placed conjugate to the microscope pupil, called pupil adaptive optics (AO). When the aberrations are spatially variant an alternative configuration involves placing the DM conjugate to the main source of aberrations, called conjugate AO. We provide a theoretical and experimental comparison of both configurations for the simplified case where spatially variant aberrations are produced by a well-defined phase screen. We pay particular attention to the resulting correction field of view (FOV). Conjugate AO is found to provide a significant FOV advantage. While this result is well known in the astronomical community, our goal here is to recast it specifically for the optical microscopy community.

Computational-Design Enabled Wearable and Tunable Metamaterials via Freeform Auxetics for Magnetic Resonance Imaging.

Metamaterials hold significant promise for enhancing the imaging capabilities of magnetic resonance imaging (MRI) machines as an additive technology, due to their unique ability to enhance local magnetic fields. However, despite their potential, the metamaterials reported in the context of MRI applications have often been impractical. This impracticality arises from their predominantly flat configurations and their susceptibility to shifts in resonance frequencies, preventing them from realizing their optimal performance. Here, a computational method for designing wearable and tunable metamaterials via freeform auxetics is introduced. The proposed computational-design tools yield an approach to solving the complex circle packing problems in an interactive and efficient manner, thus facilitating the development of deployable metamaterials configured in freeform shapes. With such tools, the developed metamaterials may readily conform to a patient's knee, ankle, head, or any part of the body in need of imaging, and while ensuring an optimal resonance frequency, thereby paving the way for the widespread adoption of metamaterials in clinical MRI applications.

A Design Approach to Reducing Stress and Distortion Caused by Adhesive Assembly in Micromachined Deformable Mirrors.

A common problem in deformable mirror assembly is that the adhesion of actuators to an optical mirror face sheet introduces unwanted topography due to large local stresses generated at the adhesive joint. A new approach to minimizing that effect is described, with inspiration taken from St. Venant's principle, a fundamental precept in solid mechanics. It is demonstrated that moving the adhesive joint to the end of a slender post extending from the face sheet largely eliminates deformation due to adhesive stresses. A practical implementation of this design innovation is described, using silicon-on-insulator wafers and deep reactive ion etching. Simulation and experiments validate the effectiveness of the approach, reducing stress-induced topography on a test structure by a factor of 50. A prototype electromagnetic DM using this design approach is described, and its actuation is demonstrated. This new design can benefit a wide range of DMs that rely on actuator arrays that are adhesively bonded to a mirror face sheet.

Computational-design Enabled Wearable and Tunable Metamaterials via Freeform Auxetics for Magnetic Resonance Imaging.

Metamaterials hold significant promise for enhancing the imaging capabilities of MRI machines as an additive technology, due to their unique ability to enhance local magnetic fields. However, despite their potential, the metamaterials reported in the context of MRI applications have often been impractical. This impracticality arises from their predominantly flat configurations and their susceptibility to shifts in resonance frequencies, preventing them from realizing their optimal performance. Here, we introduce a computational method for designing wearable and tunable metamaterials via freeform auxetics. The proposed computational-design tools yield an approach to solving the complex circle packing problems in an interactive and efficient manner, thus facilitating the development of deployable metamaterials configured in freeform shapes. With such tools, the developed metamaterials may readily conform to a patient's kneecap, ankle, head, or any part of the body in need of imaging, and while ensuring an optimal resonance frequency, thereby paving the way for the widespread adoption of metamaterials in clinical MRI applications.

High throughput screening system for engineered cardiac tissues.

Introduction: Three dimensional engineered cardiac tissues (3D ECTs) have become indispensable as in vitro models to assess drug cardiotoxicity, a leading cause of failure in pharmaceutical development. A current bottleneck is the relatively low throughput of assays that measure spontaneous contractile forces exerted by millimeter-scale ECTs typically recorded through precise optical measurement of deflection of the polymer scaffolds that support them. The required resolution and speed limit the field of view to at most a few ECTs at a time using conventional imaging. Methods: To balance the inherent tradeoff among imaging resolution, field of view and speed, an innovative mosaic imaging system was designed, built, and validated to sense contractile force of 3D ECTs seeded on a 96-well plate. Results: The system performance was validated through real-time, parallel contractile force monitoring for up to 3 weeks. Pilot drug testing was conducted using isoproterenol. Discussion: The described tool increases contractile force sensing throughput to 96 samples per measurement; significantly reduces cost, time and labor needed for preclinical cardiotoxicity assay using 3D ECT. More broadly, our mosaicking approach is a general way to scale up image-based screening in multi-well formats.

Dynamic Control of Contractile Force in Engineered Heart Tissue.

Three-dimensional engineered heart tissues (EHTs) derived from human induced pluripotent stem cells (iPSCs) have become an important resource for both drug toxicity screening and research on heart disease. A key metric of EHT phenotype is the contractile (twitch) force with which the tissue spontaneously beats. It is well-known that cardiac muscle contractility - its ability to do mechanical work - depends on tissue prestrain (preload) and external resistance (afterload). OBJECTIVES: Here, we demonstrate a technique to control afterload while monitoring contractile force exerted by EHTs. METHODS: We developed an apparatus that can regulate EHT boundary conditions using real-time feedback control. The system is comprised of a pair of piezoelectric actuators that can strain the scaffold and a microscope that can measure EHT force and length. Closed loop control allows dynamic regulation of effective EHT boundary stiffness. RESULTS: When controlled to switch instantaneously from auxotonic to isometric boundary conditions, EHT twitch force immediately doubled. Changes in EHT twitch force as a function of effective boundary stiffness were characterized and compared to twitch force in auxotonic conditions. CONCLUSION: EHT contractility can be regulated dynamically through feedback control of effective boundary stiffness. SIGNIFICANCE: The capacity to alter the mechanical boundary conditions of an engineered tissue dynamically offers a new way to probe tissue mechanics. This could be used to mimic afterload changes that occur naturally in disease, or to improve mechanical techniques for EHT maturation.

Auxetics-Inspired Tunable Metamaterials for Magnetic Resonance Imaging.

Auxetics refers to structures or materials with a negative Poisson's ratio, thereby capable of exhibiting counterintuitive behaviors. Herein, auxetic structures are exploited to design mechanically tunable metamaterials in both planar and hemispherical configurations operating at megahertz (MHz) frequencies, optimized for their application to magnetic resonance imaging (MRI). Specially, the reported tunable metamaterials are composed of arrays of interjointed unit cells featuring metallic helices, enabling auxetic patterns with a negative Poisson's ratio. The deployable deformation of the metamaterials yields an added degree of freedom with respect to frequency tunability through the resultant modification of the electromagnetic interactions between unit cells. The metamaterials are fabricated using 3D printing technology and an ≈20 MHz frequency shift of the resonance mode is enabled during deformation. Experimental validation is performed in a clinical (3.0 T) MRI system, demonstrating that the metamaterials enable a marked boost in radiofrequency field strength under resonance-matched conditions, ultimately yielding a dramatic increase in the signal-to-noise ratio (≈4.5×) of MRI. The tunable metamaterials presented herein offer a novel pathway toward the practical utilization of metamaterials in MRI, as well as a range of other emerging applications.

Three-Photon Adaptive Optics for Mouse Brain Imaging.

Three-photon microscopy (3PM) was shown to allow deeper imaging than two-photon microscopy (2PM) in scattering biological tissues, such as the mouse brain, since the longer excitation wavelength reduces tissue scattering and the higher-order non-linear excitation suppresses out-of-focus background fluorescence. Imaging depth and resolution can further be improved by aberration correction using adaptive optics (AO) techniques where a spatial light modulator (SLM) is used to correct wavefront aberrations. Here, we present and analyze a 3PM AO system for in vivo mouse brain imaging. We use a femtosecond source at 1300 nm to generate three-photon (3P) fluorescence in yellow fluorescent protein (YFP) labeled mouse brain and a microelectromechanical (MEMS) SLM to apply different Zernike phase patterns. The 3P fluorescence signal is used as feedback to calculate the amount of phase correction without direct phase measurement. We show signal improvement in the cortex and the hippocampus at greater than 1 mm depth and demonstrate close to diffraction-limited imaging in the cortical layers of the brain, including imaging of dendritic spines. In addition, we characterize the effective volume for AO correction within brain tissues, and discuss the limitations of AO correction in 3PM of mouse brain.

Nonreciprocal Magnetic Coupling Using Nonlinear Meta-Atoms.

Breaking Lorentz reciprocity is fundamental to an array of functional radiofrequency (RF) and optical devices, such as isolators and circulators. The application of external excitation, such as magnetic fields and spatial-temporal modulation, has been employed to achieve nonreciprocal responses. Alternatively, nonlinear effects may also be employed to break reciprocity in a completely passive fashion. Herein, a coupled system comprised of linear and nonlinear meta-atoms that achieves nonreciprocity based on the coupling and frequency detuning of its constituent meta-atoms is presented. An analytical model is developed based on the coupled mode theory (CMT) in order to design and optimize the nonreciprocal meta-atoms in this coupled system. Experimental demonstration of an RF isolator is performed, and the contrast between forward and backward propagation approximates 20 dB. Importantly, the use of the CMT model developed herein enables a generalizable capacity to predict the limitations of nonlinearity-based nonreciprocity, thereby facilitating the development of novel approaches to breaking Lorentz reciprocity. The CMT model and implementation scheme presented in this work may be deployed in a wide range of applications, including integrated photonic circuits, optical metamaterials, and metasurfaces, among others.

High-throughput label-free flow cytometry based on matched-filter compressive imaging.

We present a fast label-free computational flow cytometer based on a strategy of compressive imaging. Scattered light from flowing objects is sub-divided into user-defined basis patterns by a deformable mirror and routed to different detectors associated with each pattern. The patterns can be optimized to be matched to the object features of interest, thus facilitating object identification and separation. Compared to conventional scanning flow cytometers, our technique provides increased information capacity without sacrificing flow velocity. Unique features of our matched-filter strategy are that it can simultaneously probe multiple objects throughout large fields of view with long depths of field. In our proof-of-concept demonstrations, we achieve throughputs of over 10,000 particles/s, working at flow velocities of over 1m/s.

Widefield fluorescence microscopy with sensor-based conjugate adaptive optics using oblique back illumination.

We describe a wavefront sensor strategy for the implementation of adaptive optics (AO) in microscope applications involving thick, scattering media. The strategy is based on the exploitation of multiple scattering to provide oblique back illumination of the wavefront-sensor focal plane, enabling a simple and direct measurement of the flux-density tilt angles caused by aberrations at this plane. Advantages of the sensor are that it provides a large measurement field of view (FOV) while requiring no guide star, making it particularly adapted to a type of AO called conjugate AO, which provides a large correction FOV in cases when sample-induced aberrations arise from a single dominant plane (e.g., the sample surface). We apply conjugate AO here to widefield (i.e., nonscanning) fluorescence microscopy for the first time and demonstrate dynamic wavefront correction in a closed-loop implementation.

Open-loop control of a MEMS deformable mirror for large-amplitude wavefront control.

A method is introduced for predicting control voltages that will generate a prescribed surface shape on a MEMS deformable mirror. The algorithm is based upon an analytical elastic model of the mirror membrane and an empirical electromechanical model of its actuators. It is computationally simple and inherently fast. Shapes at the limit of achievable mirror spatial frequencies with up to 1.5 microm amplitudes have been achieved with less than 15 nm rms error.

In vivo fluorescent imaging of the mouse retina using adaptive optics.

In vivo imaging of the mouse retina using visible and near infrared wavelengths does not achieve diffraction-limited resolution due to wavefront aberrations induced by the eye. Considering the pupil size and axial dimension of the eye, it is expected that unaberrated imaging of the retina would have a transverse resolution of 2 microm. Higher-order aberrations in retinal imaging of human can be compensated for by using adaptive optics. We demonstrate an adaptive optics system for in vivo imaging of fluorescent structures in the retina of a mouse, using a microelectromechanical system membrane mirror and a Shack-Hartmann wavefront sensor that detects fluorescent wavefront.

A hydromechanical biomimetic cochlea: experiments and models.

The construction, measurement, and modeling of an artificial cochlea (ACochlea) are presented in this paper. An artificial basilar membrane (ABM) was made by depositing discrete Cu beams on a piezomembrane substrate. Rather than two fluid channels, as in the mammalian cochlea, a single fluid channel was implemented on one side of the ABM, facilitating the use of a laser to detect the ABM vibration on the other side. Measurements were performed on both the ABM and the ACochlea. The measurement results on the ABM show that the longitudinal coupling on the ABM is very strong. Reduced longitudinal coupling was achieved by cutting the membrane between adjacent beams using a laser. The measured results from the ACochlea with a laser-cut ABM demonstrate cochlear-like features, including traveling waves, sharp high-frequency rolloffs, and place-specific frequency selectivity. Companion computational models of the mechanical devices were formulated and implemented using a circuit simulator. Experimental data were compared with simulation results. The simulation results from the computational models of the ABM and the ACochlea are similar to their experimental counterparts.