The mechanism for directional hearing in fish.

Locating sound sources such as prey or predators is critical for survival in many vertebrates. Terrestrial vertebrates locate sources by measuring the time delay and intensity difference of sound pressure at each ear1-5. Underwater, however, the physics of sound makes interaural cues very small, suggesting that directional hearing in fish should be nearly impossible6. Yet, directional hearing has been confirmed behaviourally, although the mechanisms have remained unknown for decades. Several hypotheses have been proposed to explain this remarkable ability, including the possibility that fish evolved an extreme sensitivity to minute interaural differences or that fish might compare sound pressure with particle motion signals7,8. However, experimental challenges have long hindered a definitive explanation. Here we empirically test these models in the transparent teleost Danionella cerebrum, one of the smallest vertebrates9,10. By selectively controlling pressure and particle motion, we dissect the sensory algorithm underlying directional acoustic startles. We find that both cues are indispensable for this behaviour and that their relative phase controls its direction. Using micro-computed tomography and optical vibrometry, we further show that D. cerebrum has the sensory structures to implement this mechanism. D. cerebrum shares these structures with more than 15% of living vertebrate species, suggesting a widespread mechanism for inferring sound direction.

Ultrafast sound production mechanism in one of the smallest vertebrates.

Motion is the basis of nearly all animal behavior. Evolution has led to some extraordinary specializations of propulsion mechanisms among invertebrates, including the mandibles of the dracula ant and the claw of the pistol shrimp. In contrast, vertebrate skeletal movement is considered to be limited by the speed of muscle, saturating around 250 Hz. Here, we describe the unique propulsion mechanism by which Danionella cerebrum, a miniature cyprinid fish of only 12 mm length, produces high amplitude sounds exceeding 140 dB (re. 1 µPa, at a distance of one body length). Using a combination of high-speed video, micro-computed tomography (micro-CT), RNA profiling, and finite difference simulations, we found that D. cerebrum employ a unique sound production mechanism that involves a drumming cartilage, a specialized rib, and a dedicated muscle adapted for low fatigue. This apparatus accelerates the drumming cartilage at over 2,000 g, shooting it at the swim bladder to generate a rapid, loud pulse. These pulses are chained together to make calls with either bilaterally alternating or unilateral muscle contractions. D. cerebrum use this remarkable mechanism for acoustic communication with conspecifics.

Blazed oblique plane microscopy reveals scale-invariant inference of brain-wide population activity.

Due to the size and opacity of vertebrate brains, it has until now been impossible to simultaneously record neuronal activity at cellular resolution across the entire adult brain. As a result, scientists are forced to choose between cellular-resolution microscopy over limited fields-of-view or whole-brain imaging at coarse-grained resolution. Bridging the gap between these spatial scales of understanding remains a major challenge in neuroscience. Here, we introduce blazed oblique plane microscopy to perform brain-wide recording of neuronal activity at cellular resolution in an adult vertebrate. Contrary to common belief, we find that inferences of neuronal population activity are near-independent of spatial scale: a set of randomly sampled neurons has a comparable predictive power as the same number of coarse-grained macrovoxels. Our work thus links cellular resolution with brain-wide scope, challenges the prevailing view that macroscale methods are generally inferior to microscale techniques and underscores the value of multiscale approaches to studying brain-wide activity.

Adaptive optics for optical microscopy [Invited].

Optical microscopy is widely used to visualize fine structures. When applied to bioimaging, its performance is often degraded by sample-induced aberrations. In recent years, adaptive optics (AO), originally developed to correct for atmosphere-associated aberrations, has been applied to a wide range of microscopy modalities, enabling high- or super-resolution imaging of biological structure and function in complex tissues. Here, we review classic and recently developed AO techniques and their applications in optical microscopy.

Evolutionary divergence of locomotion in two related vertebrate species.

Locomotion exists in diverse forms in nature; however, little is known about how closely related species with similar neuronal circuitry can evolve different navigational strategies to explore their environments. Here, we investigate this question by comparing divergent swimming pattern in larval Danionella cerebrum (DC) and zebrafish (ZF). We show that DC displays long continuous swimming events when compared with the short burst-and-glide swimming in ZF. We reveal that mesencephalic locomotion maintenance neurons in the midbrain are sufficient to cause this increased swimming. Moreover, we propose that the availability of dissolved oxygen and timing of swim bladder inflation drive the observed differences in the swim pattern. Our findings uncover the neural substrate underlying the evolutionary divergence of locomotion and its adaptation to their environmental constraints.

Dynamic conjugate F-SHARP microscopy.

Optical microscopy is an indispensable tool in biomedical sciences, but its reach in deep tissues is limited due to aberrations and scattering. This problem can be overcome by wavefront-shaping techniques, albeit at limited fields of view (FOVs). Inspired by astronomical imaging, conjugate wavefront shaping can lead to an increased field of view in microscopy, but this correction is limited to a set depth and cannot be dynamically adapted. Here, we present a conjugate wavefront-shaping scheme based on focus scanning holographic aberration probing (F-SHARP). We combine it with a compact implementation that can be readily adapted to a variety of commercial and home-built two-photon microscopes. We demonstrate the power of the method by imaging with high resolution over extended FOV (>80 µm) deeper than 400 µm inside a mouse brain through a thinned skull.

Perspective on the Multiple Pathways to Changing Brain States.

In this review article, we highlight several disparate ideas that are linked to changes in brain state (i.e., sleep to arousal, Down to Up, synchronized to de-synchronized). In any discussion of the brain state, we propose that the cortical pyramidal neuron has a central position. EEG recordings, which typically assess brain state, predominantly reflect the activity of cortical pyramidal neurons. This means that the dominant rhythmic activity that characterizes a particular brain state ultimately has to manifest globally across the pyramidal neuron population. During state transitions, it is the long-range connectivity of these neurons that broadcast the resultant changes in activity to many subcortical targets. Structures like the thalamus, brainstem/hypothalamic neuromodulatory systems, and respiratory systems can also strongly influence brain state, and for many decades we have been uncovering bidirectional pathways that link these structures to state changes in the cerebral cortex. More recently, movement and active behaviors have emerged as powerful drivers of state changes. Each of these systems involve different circuits distributed across the brain. Yet, for a system-wide change in brain state, there must be a collaboration between these circuits that reflects and perhaps triggers the transition between brain states. As we expand our understanding of how brain state changes, our current challenge is to understand how these diverse sets of circuits and pathways interact to produce the changes observed in cortical pyramidal neurons.

Learned sensing: jointly optimized microscope hardware for accurate image classification.

Since its invention, the microscope has been optimized for interpretation by a human observer. With the recent development of deep learning algorithms for automated image analysis, there is now a clear need to re-design the microscope's hardware for specific interpretation tasks. To increase the speed and accuracy of automated image classification, this work presents a method to co-optimize how a sample is illuminated in a microscope, along with a pipeline to automatically classify the resulting image, using a deep neural network. By adding a "physical layer" to a deep classification network, we are able to jointly optimize for specific illumination patterns that highlight the most important sample features for the particular learning task at hand, which may not be obvious under standard illumination. We demonstrate how our learned sensing approach for illumination design can automatically identify malaria-infected cells with up to 5-10% greater accuracy than standard and alternative microscope lighting designs. We show that this joint hardware-software design procedure generalizes to offer accurate diagnoses for two different blood smear types, and experimentally show how our new procedure can translate across different experimental setups while maintaining high accuracy.

Hybrid genome assembly and annotation of Danionella translucida.

Studying neuronal circuits at cellular resolution is very challenging in vertebrates due to the size and optical turbidity of their brains. Danionella translucida, a close relative of zebrafish, was recently introduced as a model organism for investigating neural network interactions in adult individuals. Danionella remains transparent throughout its life, has the smallest known vertebrate brain and possesses a rich repertoire of complex behaviours. Here we sequenced, assembled and annotated the Danionella translucida genome employing a hybrid Illumina/Nanopore read library as well as RNA-seq of embryonic, larval and adult mRNA. We achieved high assembly continuity using low-coverage long-read data and annotated a large fraction of the transcriptome. This dataset will pave the way for molecular research and targeted genetic manipulation of this novel model organism.

Publisher Correction: Transparent Danionella translucida as a genetically tractable vertebrate brain model.

The version of this paper originally published contained errors in reference citations: in the first paragraph of the Results section, the text "This extent of optical clarity probably results from the absence of skull above the brain22. In our specimens, Nissl-stained coronal sections through the head showed that the skull surrounds the brain only laterally and ventrally" should have read "This extent of optical clarity probably results from the absence of skull above the brain21. In our specimens, Nissl-stained coronal sections through the head22 showed that the skull surrounds the brain only laterally and ventrally." In addition, the unit abbreviation "µm" was incorrectly divided at a line break in the third paragraph of the Discussion, which might have led to some confusion. These errors have been corrected in the PDF and HTML versions of the article.

Transparent Danionella translucida as a genetically tractable vertebrate brain model.

Understanding how distributed neuronal circuits integrate sensory information and generate behavior is a central goal of neuroscience. However, it has been difficult to study neuronal networks at single-cell resolution across the entire adult brain in vertebrates because of their size and opacity. We address this challenge here by introducing the fish Danionella translucida to neuroscience as a potential model organism. This teleost remains small and transparent even in adulthood, when neural circuits and behavior have matured. Despite having the smallest known adult vertebrate brain, D. translucida displays a rich set of complex behaviors, including courtship, shoaling, schooling, and acoustic communication. In order to carry out optical measurements and perturbations of neural activity with genetically encoded tools, we established CRISPR-Cas9 genome editing and Tol2 transgenesis techniques. These features make D. translucida a promising model organism for the study of adult vertebrate brain function at single-cell resolution.

Kilohertz binary phase modulator for pulsed laser sources using a digital micromirror device.

The controlled modulation of an optical wavefront is required for aberration correction, digital phase conjugation, or patterned photostimulation. For most of these applications, it is desirable to control the wavefront modulation at the highest rates possible. The digital micromirror device (DMD) presents a cost-effective solution to achieve high-speed modulation and often exceeds the speed of the more conventional liquid crystal spatial light modulator but is inherently an amplitude modulator. Furthermore, spatial dispersion caused by DMD diffraction complicates its use with pulsed laser sources, such as those used in nonlinear microscopy. Here we introduce a DMD-based optical design that overcomes these limitations and achieves dispersion-free high-speed binary phase modulation. We show that this phase modulation can be used to switch through binary phase patterns at the rate of 20 kHz in two-photon excitation fluorescence applications.

Relation between speckle decorrelation and optical phase conjugation (OPC)-based turbidity suppression through dynamic scattering media: a study on in vivo mouse skin.

Light scattering in biological tissue significantly limits the accessible depth for localized optical interrogation and deep-tissue optical imaging. This challenge can be overcome by exploiting the time-reversal property of optical phase conjugation (OPC) to reverse multiple scattering events or suppress turbidity. However, in living tissue, scatterers are highly movable and the movement can disrupt time-reversal symmetry when there is a latency in the OPC playback. In this paper, we show that the motion-induced degradation of the OPC turbidity-suppression effect through a dynamic scattering medium shares the same decorrelation time constant as that determined from speckle intensity autocorrelation - a popular conventional measure of scatterer movement. We investigated this decorrelation characteristic time through a 1.5-mm-thick dorsal skin flap of a living mouse and found that it ranges from 50 ms to 2.5 s depending on the level of immobilization. This study provides information on relevant time scales for applying OPC to living tissues.

Iterative time-reversed ultrasonically encoded light focusing in backscattering mode.

The Time-Reversed Ultrasound-Encoded (TRUE) light technique enables noninvasive focusing deep inside scattering media. However, the time-reversal procedure usually has a low signal-to-noise ratio because the intensity of ultrasound-encoded light is intrinsically low. Consequently, the contrast and resolution of TRUE focus is far from ideal, especially in the backscattering geometry, which is more practical in many biomedical applications. To improve the light intensity and resolution of TRUE focus, we developed an iterative TRUE (iTRUE) light focusing technique that employs the TRUE focus itself as a signal source (rather than diffused light) for subsequent TRUE procedures. Importantly, this iTRUE technique enables light focusing in backscattering mode. Here, we demonstrate the concept by focusing light in between scattering layers in a backscattering configuration and show that the light intensity at the focus is progressively enhanced by a factor of ~20. By scanning across a fluorescent bead between these two scattering layers, the focusing resolution in the ultrasound axial and lateral directions was improved ~2-fold and ~3-fold, respectively. We further explored the application of iTRUE in biological samples by focusing light between 1 mm thick chicken tissue and cartilage, and light intensity enhancements of the same order were also observed.

Method for auto-alignment of digital optical phase conjugation systems based on digital propagation.

Optical phase conjugation (OPC) has enabled many optical applications such as aberration correction and image transmission through fiber. In recent years, implementation of digital optical phase conjugation (DOPC) has opened up the possibility of its use in biomedical optics (e.g. deep-tissue optical focusing) due to its ability to provide greater-than-unity OPC reflectivity (the power ratio of the phase conjugated beam and input beam to the OPC system) and its flexibility to accommodate additional wavefront manipulations. However, the requirement for precise (pixel-to-pixel matching) alignment of the wavefront sensor and the spatial light modulator (SLM) limits the practical usability of DOPC systems. Here, we report a method for auto-alignment of a DOPC system by which the misalignment between the sensor and the SLM is auto-corrected through digital light propagation. With this method, we were able to accomplish OPC playback with a DOPC system with gross sensor-SLM misalignment by an axial displacement of up to~1.5 cm, rotation and tip/tilt of ~5° and in-plane displacement of ~5 mm (dependent on the physical size of the sensor and the SLM). Our auto-alignment method robustly achieved a DOPC playback peak-to-background ratio (PBR) corresponding to more than ~30 % of the theoretical maximum. As an additional advantage, the auto-alignment procedure can be easily performed at will and, as such, allows us to correct for small mechanical drifts within the DOPC systems, thus overcoming a previously major DOPC system vulnerability. We believe that this reported method for implementing robust DOPC systems will broaden the practical utility of DOPC systems.

Axial standing-wave illumination frequency-domain imaging (SWIF).

Despite their tremendous contribution to biomedical research and diagnosis, conventional spatial sampling techniques such as wide-field, point scanning or selective plane illumination microscopy face inherent limiting trade-offs between spatial resolution, field-of-view, phototoxicity and recording speed. Several of these trade-offs are the result of spatial sampling with diffracting beams. Here, we introduce a new strategy for fluorescence imaging, SWIF, which instead encodes the axial profile of a sample in the Fourier domain. We demonstrate how this can be achieved with propagation-invariant illumination patterns that extend over several millimeters and robustly propagate through layers of varying refractive index. This enabled us to image a lateral field-of-view of 0.8 mm x 1.5 mm with an axial resolution of 2.4 µm - greatly exceeding the lateral field-of-view of conventional illumination techniques (~100 µm) at comparable resolution. Thus, SWIF allowed us to surpass the limitations of diffracting illumination beams and untangle lateral field-of-view from resolution.

Model for estimating the penetration depth limit of the time-reversed ultrasonically encoded optical focusing technique.

The time-reversed ultrasonically encoded (TRUE) optical focusing technique is a method that is capable of focusing light deep within a scattering medium. This theoretical study aims to explore the depth limits of the TRUE technique for biological tissues in the context of two primary constraints - the safety limit of the incident light fluence and a limited TRUE's recording time (assumed to be 1 ms), as dynamic scatterer movements in a living sample can break the time-reversal scattering symmetry. Our numerical simulation indicates that TRUE has the potential to render an optical focus with a peak-to-background ratio of ~2 at a depth of ~103 mm at wavelength of 800 nm in a phantom with tissue scattering characteristics. This study sheds light on the allocation of photon budget in each step of the TRUE technique, the impact of low signal on the phase measurement error, and the eventual impact of the phase measurement error on the strength of the TRUE optical focus.

Focusing on moving targets through scattering samples.

Focusing light through scattering media has been a longstanding goal of biomedical optics. While wavefront shaping and optical time-reversal techniques can in principle be used to focus light across scattering media, achieving this within a scattering medium with a noninvasive and efficient reference beacon, or guide star, remains an important challenge. Here, we show optical time-reversal focusing using a new technique termed Time Reversal by Analysis of Changing wavefronts from Kinetic targets (TRACK). By taking the difference between time-varying scattering fields caused by a moving object and applying optical time reversal, light can be focused back to the location previously occupied by the object. We demonstrate this approach with discretely moved objects as well as with particles in an aqueous flow, and obtain a focal peak-to-background strength of 204 in our demonstration experiments. We further demonstrate that the generated focus can be used to noninvasively count particles in a flow-cytometry configuration-even when the particles are hidden behind a strong diffuser. By achieving optical time reversal and focusing noninvasively without any external guide stars, using just the intrinsic characteristics of the sample, this work paves the way to a range of scattering media imaging applications, including underwater and atmospheric focusing as well as noninvasive in vivo flow cytometry.

Physical key-protected one-time pad.

We describe an encrypted communication principle that forms a secure link between two parties without electronically saving either of their keys. Instead, random cryptographic bits are kept safe within the unique mesoscopic randomness of two volumetric scattering materials. We demonstrate how a shared set of patterned optical probes can generate 10 gigabits of statistically verified randomness between a pair of unique 2 mm(3) scattering objects. This shared randomness is used to facilitate information-theoretically secure communication following a modified one-time pad protocol. Benefits of volumetric physical storage over electronic memory include the inability to probe, duplicate or selectively reset any bits without fundamentally altering the entire key space. Our ability to securely couple the randomness contained within two unique physical objects can extend to strengthen hardware required by a variety of cryptographic protocols, which is currently a critically weak link in the security pipeline of our increasingly mobile communication culture.

Speckle-scale focusing in the diffusive regime with time-reversal of variance-encoded light (TROVE).

Focusing of light in the diffusive regime inside scattering media has long been considered impossible. Recently, this limitation has been overcome with time reversal of ultrasound-encoded light (TRUE), but the resolution of this approach is fundamentally limited by the large number of optical modes within the ultrasound focus. Here, we introduce a new approach, time reversal of variance-encoded light (TROVE), which demixes these spatial modes by variance-encoding to break the resolution barrier imposed by the ultrasound. By encoding individual spatial modes inside the scattering sample with unique variances, we effectively uncouple the system resolution from the size of the ultrasound focus. This enables us to demonstrate optical focusing and imaging with diffuse light at unprecedented, speckle-scale lateral resolution of ~ 5 µm.