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.

Dual-update data-driven control of deformable mirrors using Walsh basis functions.

We develop a novel data-driven method for deformable mirror (DM) control. The developed method updates both the DM model and DM control actions that produce desired mirror surface shapes. The novel method explicitly takes into account actuator constraints and couples a feedback-control algorithm with an algorithm for recursive estimation of DM influence function models. We also explore the possibility of using Walsh basis functions for DM control. By expressing the desired and observed mirror surface shapes as sums of Walsh pattern matrices, we formulate the control problem in the 2D Walsh basis domain. We thoroughly experimentally verify the developed approach on a 140-actuator MEMS DM, developed by Boston Micromachines. Our results show that the novel method produces the root-mean-square surface error in the 14-40 nanometer range. These results can additionally be improved by tuning the control and estimation parameters. The developed approach is also applicable to other DM types such as segmented DMs.

General approach to precise deformable mirror control.

We develop a simple and effective control method for accurate control of deformable mirrors (DMs). For a desired DM surface profile and using batches of observed surface profile data, the proposed method adaptively determines both a DM model (influence matrix) and control actions that produce the desired surface profile with good accuracy. In the first iteration, the developed method estimates a DM influence matrix by solving a multivariable least-squares problem. This matrix is then used to compute the control actions by solving a constrained least-squares problem. Then, the computed actions are randomly perturbed and applied to the DM to generate a new batch of surface profile data. The new data batch is used to estimate a new influence matrix that is then used to re-compute control actions. This procedure is repeated until convergence is achieved. The method is experimentally tested on a Boston Micromachines DM with 140 micro-electronic-mechanical-system actuators. Our experimental results show that the developed control approach can achieve accurate correction despite significant DM nonlinearities. Using only a few control iterations, the developed method is able to produce a surface profile root-mean-square error that varies from 5 - 30 [nm] for most of the tested Zernike wave-front modes without using direct feedback control. These results can additionally be improved by using larger data batches and more iterations or by combining the developed approach with feedback control. Finally, as we experimentally demonstrate, the developed method can be used to estimate a DM model that can effectively be used for a single-step open-loop DM control.

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.

Volumetric chemical imaging in vivo by a remote-focusing stimulated Raman scattering microscope.

Operable under ambient light and providing chemical selectivity, stimulated Raman scattering (SRS) microscopy opens a new window for imaging molecular events on a human subject, such as filtration of topical drugs through the skin. A typical approach for volumetric SRS imaging is through piezo scanning of an objective lens, which often disturbs the sample and offers a low axial scan rate. To address these challenges, we have developed a deformable mirror-based remote-focusing SRS microscope, which not only enables high-quality volumetric chemical imaging without mechanical scanning of the objective but also corrects the system aberrations simultaneously. Using the remote-focusing SRS microscope, we performed volumetric chemical imaging of living cells and captured in real time the dynamic diffusion of topical chemicals into human sweat pores.

Extended depth-of-field microscopy with a high-speed deformable mirror.

We present a wide-field fluorescence microscopy add-on that provides a fast, light-efficient extended depth-of-field (EDOF) using a deformable mirror with an update rate of 20 kHz. Out-of-focus contributions in the raw EDOF images are suppressed with a deconvolution algorithm derived directly from the microscope 3D optical transfer function. Demonstrations of the benefits of EDOF microscopy are shown with GCaMP-labeled mouse brain tissue.

Dual fluorescence-absorption deconvolution applied to extended-depth-of-field microscopy.

Fast imaging over large volumes can be obtained in a simple manner with extended-depth-of-field (EDOF) microscopy. A standard technique of Wiener deconvolution can correct for the blurring inherent in EDOF images. We compare Wiener deconvolution with an alternative, parameter-free technique based on the dual reconstruction of fluorescence and absorption layers in a sample. This alternative technique provides significantly enhanced reconstruction contrast owing to a quadratic positivity constraint that intrinsically favors sparse solutions. We demonstrate the advantages of this technique with mouse neuronal images acquired in vivo.

Axial range of conjugate adaptive optics in two-photon microscopy.

We describe an adaptive optics technique for two-photon microscopy in which the deformable mirror used for aberration compensation is positioned in a plane conjugate to the plane of the aberration. We demonstrate in a proof-of-principle experiment that this technique yields a large field of view advantage in comparison to standard pupil-conjugate adaptive optics. Further, we show that the extended field of view in conjugate AO is maintained over a relatively large axial translation of the deformable mirror with respect to the conjugate plane. We conclude with a discussion of limitations and prospects for the conjugate AO technique in two-photon biological microscopy.

Axial range of conjugate adaptive optics in two-photon microscopy: erratum.

We correct an omission from the Acknowledgments section of our manuscript.

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.

Focusing polychromatic light through strongly scattering media.

We demonstrate feedback-optimized focusing of spatially coherent polychromatic light after transmission through strongly scattering media, and describe the relationship between optimized focus intensity and initial far-field speckle contrast. Optimization is performed using a MEMS spatial light modulator with camera-based or spectrometer-based feedback. We observe that the spectral bandwidth of the optimized focus depends on characteristics of the feedback signal. We interpret this dependence as a modification in the number of independent frequency components, or spectral correlations, transmitted by the sample, and introduce a simple model for polychromatic focus enhancement that is corroborated by experiment with calibrated samples.

Aberration compensation in aplanatic solid immersion lens microscopy.

The imaging quality of an aplanatic SIL microscope is shown to be significantly degraded by aberrations, especially when the samples have thicknesses that are more than a few micrometers thicker or thinner than the design thickness. Aberration due to the sample thickness error is modeled and compared with measurements obtained in a high numerical aperture (NA ~3.5) microscope. A technique to recover near-ideal imaging quality by compensating aberrations using a MEMS deformable mirror is described and demonstrated.

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.

Vector transmission matrix for the polarization behavior of light propagation in highly scattering media.

Recently the optical transmission matrix (TM) has been shown to be useful in controlling the propagation of light in highly scattering media. In this paper, we present the vector transmission matrix (VTM) which, unlike the TM, captures both the intensity and polarization transmission property of the scattering medium. We present an experimental technique for measuring the absolute values of the VTM elements which is in contrast to existing techniques whereby the TM elements are measured to within a scaling factor. The usefulness of the VTM is illustrated by showing that it can be used to both predict and control the magnitude of the complex polarization ratio of the light focused through the scattering medium. To the best of our knowledge, this is the first study to show the possibility of controlling the polarization of the light transmitted through highly scattering media.