Machine learning approach for ambient-light-corrected parameters and the Pupil Reactivity (PuRe) score in smartphone-based pupillometry.

Introduction: The pupillary light reflex (PLR) is the constriction of the pupil in response to light. The PLR in response to a pulse of light follows a complex waveform that can be characterized by several parameters. It is a sensitive marker of acute neurological deterioration, but is also sensitive to the background illumination in the environment in which it is measured. To detect a pathological change in the PLR, it is therefore necessary to separate the contributions of neuro-ophthalmic factors from ambient illumination. Illumination varies over several orders of magnitude and is difficult to control due to diurnal, seasonal, and location variations. Methods and results: We assessed the sensitivity of seven PLR parameters to differences in ambient light, using a smartphone-based pupillometer (AI Pupillometer, Solvemed Inc.). Nine subjects underwent 345 measurements in ambient conditions ranging from complete darkness (<5 lx) to bright lighting (≲10,000 lx). Lighting most strongly affected the initial pupil size, constriction amplitude, and velocity. Nonlinear models were fitted to find the correction function that maximally stabilized PLR parameters across different ambient light levels. Next, we demonstrated that the lighting-corrected parameters still discriminated reactive from unreactive pupils. Ten patients underwent PLR testing in an ophthalmology outpatient clinic setting following the administration of tropicamide eye drops, which rendered the pupils unreactive. The parameters corrected for lighting were combined as predictors in a machine learning model to produce a scalar value, the Pupil Reactivity (PuRe) score, which quantifies Pupil Reactivity on a scale 0-5 (0, non-reactive pupil; 0-3, abnormal/"sluggish" response; 3-5, normal/brisk response). The score discriminated unreactive pupils with 100% accuracy and was stable under changes in ambient illumination across four orders of magnitude. Discussion: This is the first time that a correction method has been proposed to effectively mitigate the confounding influence of ambient light on PLR measurements, which could improve the reliability of pupillometric parameters both in pre-hospital and inpatient care settings. In particular, the PuRe score offers a robust measure of Pupil Reactivity directly applicable to clinical practice. Importantly, the formulae behind the score are openly available for the benefit of the clinical research community.

Noise-resistant phase imaging with intensity correlation.

Interferometric methods form the basis of highly sensitive measurement techniques from astronomy to bioimaging. Interferometry typically requires high stability between the measured and reference beams. The presence of rapid phase fluctuations washes out interference fringes, making phase profile recovery impossible. This challenge can be addressed by shortening the measurement time. However, such an approach reduces photon-counting rates, precluding applications in low-intensity imaging. We introduce a phase imaging technique which is immune to time-dependent phase fluctuations. Our technique, relying on intensity correlation instead of direct intensity measurements, allows one to obtain high interference visibility for arbitrarily long acquisition times. We prove the optimality of our method using the Cramér-Rao bound in the extreme case when no more than two photons are detected within the time window of phase stability. Our technique will broaden prospects in phase measurements, including emerging applications such as in infrared and x-ray imaging and quantum and matter-wave interferometry.

Dual-polarity voltage imaging of the concurrent dynamics of multiple neuron types.

Genetically encoded fluorescent voltage indicators are ideally suited to reveal the millisecond-scale interactions among and between targeted cell populations. However, current indicators lack the requisite sensitivity for in vivo multipopulation imaging. We describe next-generation green and red voltage sensors, Ace-mNeon2 and VARNAM2, and their reverse response-polarity variants pAce and pAceR. Our indicators enable 0.4- to 1-kilohertz voltage recordings from >50 spiking neurons per field of view in awake mice and ~30-minute continuous imaging in flies. Using dual-polarity multiplexed imaging, we uncovered brain state-dependent antagonism between neocortical somatostatin-expressing (SST+) and vasoactive intestinal peptide-expressing (VIP+) interneurons and contributions to hippocampal field potentials from cell ensembles with distinct axonal projections. By combining three mutually compatible indicators, we performed simultaneous triple-population imaging. These approaches will empower investigations of the dynamic interplay between neuronal subclasses at single-spike resolution.

Fundamental bounds on the fidelity of sensory cortical coding.

How the brain processes information accurately despite stochastic neural activity is a longstanding question1. For instance, perception is fundamentally limited by the information that the brain can extract from the noisy dynamics of sensory neurons. Seminal experiments2,3 suggest that correlated noise in sensory cortical neural ensembles is what limits their coding accuracy4-6, although how correlated noise affects neural codes remains debated7-11. Recent theoretical work proposes that how a neural ensemble's sensory tuning properties relate statistically to its correlated noise patterns is a greater determinant of coding accuracy than is absolute noise strength12-14. However, without simultaneous recordings from thousands of cortical neurons with shared sensory inputs, it is unknown whether correlated noise limits coding fidelity. Here we present a 16-beam, two-photon microscope to monitor activity across the mouse primary visual cortex, along with analyses to quantify the information conveyed by large neural ensembles. We found that, in the visual cortex, correlated noise constrained signalling for ensembles with 800-1,300 neurons. Several noise components of the ensemble dynamics grew proportionally to the ensemble size and the encoded visual signals, revealing the predicted information-limiting correlations12-14. Notably, visual signals were perpendicular to the largest noise mode, which therefore did not limit coding fidelity. The information-limiting noise modes were approximately ten times smaller and concordant with mouse visual acuity15. Therefore, cortical design principles appear to enhance coding accuracy by restricting around 90% of noise fluctuations to modes that do not limit signalling fidelity, whereas much weaker correlated noise modes inherently bound sensory discrimination.

RecV recombinase system for in vivo targeted optogenomic modifications of single cells or cell populations.

Brain circuits comprise vast numbers of interconnected neurons with diverse molecular, anatomical and physiological properties. To allow targeting of individual neurons for structural and functional studies, we created light-inducible site-specific DNA recombinases based on Cre, Dre and Flp (RecVs). RecVs can induce genomic modifications by one-photon or two-photon light induction in vivo. They can produce targeted, sparse and strong labeling of individual neurons by modifying multiple loci within mouse and zebrafish genomes. In combination with other genetic strategies, they allow intersectional targeting of different neuronal classes. In the mouse cortex they enable sparse labeling and whole-brain morphological reconstructions of individual neurons. Furthermore, these enzymes allow single-cell two-photon targeted genetic modifications and can be used in combination with functional optical indicators with minimal interference. In summary, RecVs enable spatiotemporally precise optogenomic modifications that can facilitate detailed single-cell analysis of neural circuits by linking genetic identity, morphology, connectivity and function.

Kilohertz two-photon brain imaging in awake mice.

Two-photon microscopy is a mainstay technique for imaging in scattering media and normally provides frame-acquisition rates of ~10-30 Hz. To track high-speed phenomena, we created a two-photon microscope with 400 illumination beams that collectively sample 95,000-211,000 µm2 areas at rates up to 1 kHz. Using this microscope, we visualized microcirculatory flow, fast venous constrictions and neuronal Ca2+ spiking with millisecond-scale timing resolution in the brains of awake mice.

High-Capacity Angularly Multiplexed Holographic Memory Operating at the Single-Photon Level.

We experimentally demonstrate an angularly multiplexed holographic memory capable of intrinsic generation, storage, and retrieval of multiple photons, based on an off-resonant Raman interaction in warm rubidium-87 vapors. The memory capacity of up to 60 independent atomic spin-wave modes is evidenced by analyzing angular distributions of coincidences between Stokes and time-delayed anti-Stokes light, observed down to the level of single spin-wave excitation during the several-microsecond memory lifetime. We also propose how to practically enhance rates of single- and multiple-photon generation by combining our multimode emissive memory with existing fast optical switches.

Mode engineering for realistic quantum-enhanced interferometry.

Quantum metrology overcomes standard precision limits by exploiting collective quantum superpositions of physical systems used for sensing, with the prominent example of non-classical multiphoton states improving interferometric techniques. Practical quantum-enhanced interferometry is, however, vulnerable to imperfections such as partial distinguishability of interfering photons. Here we introduce a method where appropriate design of the modal structure of input photons can alleviate deleterious effects caused by another, experimentally inaccessible degree of freedom. This result is accompanied by a laboratory demonstration that a suitable choice of spatial modes combined with position-resolved coincidence detection restores entanglement-enhanced precision in the full operating range of a realistic two-photon Mach-Zehnder interferometer, specifically around a point which otherwise does not even attain the shot-noise limit due to the presence of residual distinguishing information in the spectral degree of freedom. Our method highlights the potential of engineering multimode physical systems in metrologic applications.

Shot-by-shot imaging of Hong-Ou-Mandel interference with an intensified sCMOS camera.

We report the first observation of Hong-Ou-Mandel (HOM) interference of highly indistinguishable photon pairs with spatial resolution. Direct imaging of two-photon coalescence with an intensified sCMOS camera system clearly reveals spatially separated photons appearing pairwise within one of the two modes. With the use of the camera system, we quantified the number of pairs and recovered the full HOM dip yielding 96.3% interference visibility, as well as counted the number of coalesced pairs. We retrieved the spatial modes of both interfering photons by performing a proof-of-principle demonstration of a new, low-noise, high-resolution coincidence imaging scheme.

Hamiltonian design in readout from room-temperature Raman atomic memory.

We present an experimental demonstration of the Hamiltonian manipulation in light-atom interface in Raman-type warm rubidium-87 vapor atomic memory. By adjusting the detuning of the driving beam we varied the relative contributions of the Stokes and anti-Stokes scattering to the process of four-wave mixing which reads out a spatially multimode state of atomic memory. We measured the temporal evolution of the readout fields and the spatial intensity correlations between write-in and readout as a function of detuning with the use of an intensified camera. The correlation maps enabled us to resolve between the anti-Stokes and the Stokes scattering and to quantify their contributions. Our experimental results agree quantitatively with a simple, plane-wave theoretical model we provide. They allow for a simple interpretation of the coaction of the anti-Stokes and the Stokes scattering at the readout stage. The Stokes contribution yields additional, adjustable gain at the readout stage, albeit with inevitable extra noise. Here we provide a simple and useful framework to trace it and the results can be utilized in the existing atomic memories setups. Furthermore, the shown Hamiltonian manipulation offers a broad range of atom-light interfaces readily applicable in current and future quantum protocols with atomic ensembles.

High-fidelity spatially resolved multiphoton counting for quantum imaging applications.

We present a method for spatially resolved multiphoton counting based on an intensified camera with the retrieval of multimode photon statistics fully accounting for nonlinearities in the detection process. The scheme relies on one-time quantum tomographic calibration of the detector. Faithful, high-fidelity reconstruction of single- and two-mode statistics of multiphoton states is demonstrated for coherent states and their statistical mixtures. The results consistently exhibit classical values of the Mandel parameter and the noise reduction factor in contrast to raw statistics of camera photo-events. Detector operation is reliable for illumination levels up to the average of one detected photon per an event area-substantially higher than in previous approaches to characterize quantum statistical properties of light with spatial resolution.

Generation and delayed retrieval of spatially multimode Raman scattering in warm rubidium vapors.

We apply collective Raman scattering to create, store and retrieve spatially multimode light in warm rubidium-87 vapors. The light is created in a spontaneous Stokes scattering process. This is accompanied by the creation of counterpart collective excitations in the atomic ensemble - the spin waves. After a certain storage time we coherently convert the spin waves into the light in deterministic anti-Stokes scattering. The whole process can be regarded as a delayed four-wave mixing which produces pairs of correlated, delayed random images. Storage of higher order spatial modes up to microseconds is possible owing to usage of a buffer gas. We study the performance of the Raman scattering, storage and retrieval of collective excitations focusing on spatial effects and the influence of decoherence caused by diffusion of rubidium atoms in different buffer gases. We quantify the number of modes created and retrieved by analyzing statistical correlations of intensity fluctuations between portions of the light scattered in the far field.