The possibility of using light to image and manipulate neuronal activity, at the heart of Neurophotonics, has provided new irreplaceable tools to study brain function. In particular, the combination of multiphoton microscopy and optogenetics allows researchers to interact with neuronal circuits with single-cell resolution in living brain tissues. However, significant optical challenges remain to empower new discoveries in Neuroscience. This Review focuses on three critical areas for future development: (1) expanding imaging and optogenetic stimulation to larger fields of view and faster acquisition speeds, while maintaining single-cell resolution and minimizing photodamage; (2) enabling access to deeper brain regions to study currently inaccessible neuronal circuits; and (3) developing optical techniques for studying natural behaviors in freely moving animals. For each of these challenges, we review the current state-of-the-art and suggest future directions with the potential to transform the field.

Fluorescence microscopes can record the dynamics of living cells with high spatio-temporal resolution in a single plane. However, monitoring rapid and dim fluorescence fluctuations, e.g induced by neuronal activity in the brain, remains challenging for 3D-distributed emitters due to out-of-focus fluorescence background, a restricted photon budget, and the speed limit of conventional scanning systems. Here, we introduce a Thermally Adaptive Surface strategy, capable of simultaneously recording, at camera framerate, the activity of 3D-distributed objects. This innovative microscope leverages on an array of thermally tuneable microlenses that offer low chromatic aberration and high transmission, and can be combined with patterned illumination to provide optical sectioning. We demonstrate its potential in vivo, by simultaneously monitoring fast fluorescent dynamics at different depths in the zebrafish larval brain, at a rate of 0.5 kHz and over a large field of view (360um x 360um).

Single-shot hyperspectral wavefront sensing is essential for applications like spatio-spectral coupling metrology in high-power laser or fast material dispersion imaging. Under broadband illumination, traditional wavefront sensors assume an achromatic wavefront, which makes them unsuitable. We introduce a hyperspectral wavefront sensing scheme based on the Hartmann wavefront sensing principles, employing a multicore fiber as a Hartmann mask to overcome these limitations. Our system leverages the angular memory effect and limited spectral correlation width of the multicore fiber, encoding wavefront gradients into displacements and the spectral information into uncorrelated speckle patterns. This method retains the simplicity, compactness, and single-shot capability of conventional wavefront sensors, with only a slight increase in computational complexity. It also allows a tunable trade-off between spatial and spectral resolution. We demonstrate its efficacy for recording the hyperspectral wavefront cube from single-pulse acquisitions at the Apollon multi-petawatt laser facility, and for performing multispectral microscopic imaging of dispersive phase objects. The authors introduce a high-resolution and spectrally-resolved optical wavefront sensor based on a multicore fiber. Capturing information in one single shot, this instrument advances ultrafast laser metrology and quantitative dispersion microscopy.

We recently proposed a new wavefront shaping concept, coined as Smartlens, in which the phase of the transmitted light is shaped by engineering the temperature landscape in a thermo-optical material. Individually or in arrays, these microscale devices can generate complex functions based on either pure, or a combination of, Zernike polynomials, including lenses or electrically-tuneable aberration correctors. We will see how this concept could complement the existing optical shaping toolbox by offering low-chromatic-aberration, polarization-insensitive and transmission-mode micro-components. I will then delve into the potential of a single reconfigurable Smartlens to embed adaptive optics within a fluorescence endoscope. Finally, I will show that a Smartlens Array can enable multiplane Ca2+ or voltage imaging in Zebrafish at KHz frame rates.

In astronomy or biological imaging, refractive index inhomogeneities of e.g. atmosphere or tissues induce optical aberrations which degrade the desired information hidden behind the medium. A standard approach consists in measuring these aberrations with a wavefront sensor (e.g Shack-Hartmann) located in the pupil plane, and compensating them either digitally or by adaptive optics with a wavefront shaper. However, in its usual implementation this strategy can only extract aberrations within a single isoplanatic patch, i.e. a region where the aberrations remain correlated. This limitation severely reduces the effective field-of-view in which the correction can be performed. Here, we propose a new wavefront sensing method capable of measuring, in a single shot, various pupil aberrations corresponding to multiple isoplanatic patches. The method, based on a thin diffuser (i.e a random phase mask), exploits the dissimilarity between different speckle regions to multiplex several wavefronts incoming from various incidence angles. We present proof-of-concept experiments carried out in wide-field fluorescence microscopy. A digital deconvolution procedure in each isoplanatic patch yields accurate aberration correction within an extended field-of-view. This approach is of interest for adaptive optics applications as well as diffractive optical tomography.

Aberrations and multiple scattering in biological tissues critically distort light beams into highly complex speckle patterns. In this regard, digital optical phase conjugation (DOPC) is a promising technique enabling in-depth focusing. However, DOPC becomes challenging when using fluorescent guide stars for four main reasons: the low photon budget available, the large spectral bandwidth of the fluorescent signal, the Stokes shift between the emission and the excitation wavelength, and the absence of reference beam preventing holographic measurement. Here, we demonstrate the possibility to focus a laser beam through multiple-scattering samples by measuring speckle fields in a single acquisition step with a reference-free, high-resolution wavefront sensor. By taking advantage of the large spectral bandwidth of forward multiply scattering samples, digital fluorescence phase conjugation is achieved to focus a laser beam at the excitation wavelength while measuring the broadband speckle field arising from a micrometer-sized fluorescent bead.

Many processes in microfluidics and biology are driven or affected by viscosity. While several methods are able to measure this parameter globally, very few can provide high resolution viscosity images. Optimizing the locality of viscosity measurements demands smaller probes but also shorter lateral diffusion lengths and measurement times. Here, we propose to use sub-micrometer magnetic rods to perform high resolution viscosity imaging. An external magnetic field forces the oscillation of superparamagnetic iron oxide rods. Under linearly polarized illumination, the rotation of these highly anisotropic optical scatterers induces a blinking which is analyzed by heterodyne holography. The spectral analysis of the rotation dynamics yields a regime transition frequency from which the local viscosity is deduced. Holography provides a 3D optical field reconstruction and 3D superlocalization of the rods, which allows super-resolved viscosity measurements. Relying on the fast Brownian rotation instead of the slower translation component of nanorods therefore allows faster measurements and, crucially, smaller effective voxels for viscosity determination. We thus demonstrate that viscosity imaging is possible with a 0.5 (micron)^3 3D-resolution.

As miniaturization becomes a growing trend in optical systems, the ability to precisely manipulate wavefronts within micrometric pupils becomes crucial. Extensive efforts to develop integrated micro-optics primarily led to tunable microlenses. Among these approaches, SmartLenses, which use predesigned microheaters to locally change the refractive index in a transparent thermo-optical material, allow to produce tunable micro-optics with free-form shape. However, the shape and sign of the generated wavefront profile are fixed, predetermined by the geometry of the resistor, which severely limits its use, e.g., for aberration correction. Here, we report a precise reconfigurability of the generated wavefront through dynamic shaping of the temperature distribution, enabled by an independent control of concentric resistors. As a proof of principle, we demonstrate a bimodal SmartLens that simultaneously acts as a converging/diverging lens and a positive/negative spherical aberration corrector. Through independent control of Zernike modes, this approach paves the way for compact, broadband, transparent and polarization-insensitive wavefront shapers, with a broad range of potential applications, from endoscopy to information technology.

We demonstrate the ability to perform single-shot digital optical phase conjugation from a Stokes shifted fluorescent incoherent guide stars hidden behind a forward scattering sample with a high-resolution wavefront sensor.

Single-shot hyperspectral wavefront sensing is essential for applications like spatio-spectral coupling metrology in high power laser or fast material dispersion imaging. Under broadband illumination, traditional wavefront sensors assume an achromatic wavefront, which makes them unsuitable. We introduce a hyperspectral wavefront sensing scheme based on the Hartmann wavefront sensing principles, employing a multicore fiber as a modified Hartmann mask to overcome these limitations. Our system leverages the angular memory effect and spectral decorrelation from the multicore fiber, encoding wavefront gradients into displacements and the spectral information into uncorrelated patterns. This method retains the simplicity, compactness, and single-shot capability of conventional wavefront sensors, with only a slight increase in computational complexity. It also allows a tunable trade-off between spatial and spectral resolution. We demonstrate its efficacy for recording the hyperspectral wavefront cube from single-pulse acquisitions at the Apollon multi-PW laser facility, and for performing multispectral microscopic imaging of dispersive phase objects.

Quantitative phase imaging enables precise and label-free characterizations of individual nano-objects within a large volume, without a priori knowledge of the sample or imaging system. While emerging common path implementations are simple enough to promise a broad dissemination, their phase sensitivity still falls short of precisely estimating the mass or polarizability of vesicles, viruses, or nanoparticles in single-shot acquisitions. In this paper, we revisit the Zernike filtering concept, originally crafted for intensity-only detectors, with the aim of adapting it to wavefront imaging. We demonstrate, through numerical simulation and experiments based on high-resolution wavefront sensing, that a simple Fourier-plane add-on can significantly enhance phase sensitivity for subdiffraction objects & horbar;achieving over an order of magnitude increase (x12)& horbar;while allowing the quantitative retrieval of both intensity and phase. This advancement allows for more precise nano-object detection and metrology.

Studying networks of interconnected neurons located in different planes at physiological speed is essential to understand the brain. However, 3D calcium or voltage imaging remain challenging since dim fluorescence signals located in non-deterministic 3D (x, y, z) positions need to be imaged at a high frame rates (typ. 0.1-1 KHz) with a good signal-to-noise ratio to detect short action potentials (typ. 1 ms for voltage imaging) [1], [2]. When imaging such short, 3D-distributed and dim events, the main drawbacks of sequential z-scanning of the imaging plane are the reduction of SNR, temporal resolution, and an increased risk of missing action potentials occurring in a given plane while imaging another one.

Monitoring chemical reactions in solutions at the scale of individual entities is challenging: single-particle detection requires small confocal volumes, which are hardly compatible with Brownian motion, particularly when long integration times are necessary. Here, we propose a real-time (10 Hz) holography-based nm-precision 3D tracking of single moving nanoparticles. Using this localization, the confocal collection volume is dynamically adjusted to follow the moving nanoparticle and allow continuous spectroscopic monitoring. This concept is applied to study galvanic exchange in freely moving colloidal silver nanoparticles with gold ions generated in situ. While the Brownian trajectory reveals particle size, spectral shifts dynamically reveal composition changes and transformation kinetics at the single-object level, pointing at different transformation kinetics for free and tethered particles.

Long wave infrared (LWIR) radiation (7-14 μm) allows illumination-less imaging, and spectroscopic chemical identification. Infrared imaging is ubiquitous in defense thermography, airborne and atmospheric sensing, fault detection, and medical testing. Visible speckle imaging can successfully image through complex scattering media. We describe a novel broadband LWIR speckle imaging-based wavefront sensor, utilizing a thin diffuser with an uncooled microbolometric camera. Due to the thin diffuser, local phase gradients produce speckle deformations which are estimated by a rapid image registration algorithm to generate a phase gradient map, whose 2-D integration yields the reconstructed wavefront. We demonstrate LWIR wavefront reconstruction using our setup in infrared optical samples, with future applications for LWIR imaging through visually non-transparent materials.

We report on the use of a thin diffuser placed in the close vicinity of a camera sensor as a simple and effective way to superlocalize plasmonic nanoparticles in 3D. This method is based on holographic reconstruction via quantitative phase and intensity measurements of a light field after its interaction with nanoparticles. We experimentally demonstrate that this thin diffuser can be used as a simple add-on to a standard bright-field microscope to allow the localization of 100 nm gold nanoparticles at video rate with nanometer precision (1.3 nm laterally and 6.3 nm longitudinally). We exemplify the approach by revealing the dynamic Brownian trajectory of a gold nanoparticle trapped in various pockets within an agarose gel. The proposed method provides a simple but highly performant way to track nanoparticles in 3D.

A novel broadband infrared (IR) speckle imaging system with a thin scatterer and an uncooled microbolometric camera is employed to encode wavefront phase variations as local speckle deformations. The phase reconstruction from speckle shifts using a fast diffeomorphic algorithm ultimately demonstrates IR wavefront reconstruction through complex media.

Individual nanoparticle spectroscopic characterization is fundamental, but challenging in liquids. While confocal selectivity is necessary to isolate a particle in a crowd, Brownian motion constantly offsets the particle from the light collection volume. Here, we present a system able to acquire holograms and reconstruct them to precisely determine the 3D position of a particle in real time. These coordinates drive an adaptive system comprising two galvanometric mirrors (x,y, transverse directions) and a tunable lens (z, longitudinal) which redirect light scattered from the corresponding region of space towards the confocal entrance of a spectrometer, thus allowing long spectral investigations on individual, freely-moving particles. A study of the movements and spectra of individual 100 nm Au nanoparticles undergoing two types of aggregations illustrates the possibilities of the method.