In biological microscopy, light scattering represents the main limitation to image at depth. Recently, a set of wavefront shaping techniques has been developed in order to manipulate coherent light in strongly disordered materials. The Transmission Matrix approach has shown its capability to inverse the effect of scattering and efficiently focus light. In practice, the matrix is usually measured using an invasive detector or low-resolution acoustic guide stars. Here, we introduce a non-invasive and all-optical strategy based on linear fluorescence to reconstruct the transmission matrices, to and from a fluorescent object placed inside a scattering medium. It consists in demixing the incoherent patterns emitted by the object using low-rank factorizations and phase retrieval algorithms. We experimentally demonstrate the efficiency of this method through robust and selective focusing. Additionally, from the same measurements, it is possible to exploit memory effect correlations to image and reconstruct extended objects. This approach opens up a new route towards imaging in scattering media with linear or non-linear contrast mechanisms.
Fluorescence is a powerful mean to probe information processing in the mammalian brain. However, neuronal tissues are highly heterogeneous and thus opaque to light. A wide set of non-invasive or invasive techniques for scattered light rejection, optical sectioning or localized excitation, have been developed, but non-invasive optical recording of activity through highly scattering layer beyond the ballistic regime is to date impossible. Here, we show that functional signals from fluorescent time-varying sources located below an highly scattering tissue can be retrieved efficiently, by exploiti…
Non-invasive optical focusing inside scattering media is still a big challenge because inhomogeneous media scatter both incoming photons for focusing and outgoing photons for observation. Various approaches, utilizing non-linear fluorescence or ultrasound, have been reported to address this difficulty. However, implementation of these methods is complicated and highly expensive, as ultrafast laser systems or photo-acoustic equipment must be employed. Here, we demonstrate a wavefront shaping technique to achieve non-invasive focusing (NiF) inside scattering media using only a linear fluorescent…
A major open challenge in neuroscience is the ability to measure and perturb neural activity in vivo from well-defined neural sub-populations at cellular resolution anywhere in the brain. However, limitations posed by scattering and absorption prohibit non-invasive (surface) multiphoton approaches for deep (>2mm) structures, while Gradient Refreactive Index (GRIN) endoscopes are thick and cause significant damage upon insertion. Here, we demonstrate a novel microendoscope to image neural activity at arbitrary depths via an ultrathin multimode optical fiber (MMF) probe that is 5-10X thinner tha…
Computational Cannula Microscopy (CCM) is a high-resolution widefield fluorescence imaging approach deep inside tissue, which is minimally invasive. Rather than using conventional lenses, a surgical cannula acts as a lightpipe for both excitation and fluorescence emission, where computational methods are used for image visualization. Here, we enhance CCM with artificial neural networks to enable 3D imaging of cultured neurons and fluorescent beads, the latter inside a volumetric phantom. We experimentally demonstrate transverse resolution of ~6um, field of view ~200um and axial sectioning of ~…
Optical imaging deep inside scattering media remains a fundamental problem in bio-imaging. While wavefront shaping has been shown to allow focusing of coherent light at depth, achieving it non-invasively remains a challenge. Various feedback mechanisms, in particular acoustic or non-linear fluorescence-based, have been put forward for this purpose. Non-invasive focusing at depth on fluorescent objects with linear excitation is, however, still unresolved. Here we report a simple method for focusing inside a scattering medium in an epi-detection geometry with a linear signal: optimizing the spat…
Time-resolved fluorescence imaging is a key tool in biomedical applications, as it allows to non-invasively obtain functional and structural information. However, the big amount of collected data introduces challenges in both acquisition speed and processing needs. Here, we introduce a novel technique that allows to reconstruct a Giga-voxel 4D hypercube in a fast manner while only measuring 0.03 % of the information. The system combines two single-pixel cameras and a conventional 2D array detector working in parallel. Data fusion techniques are introduced to combine the individual 2D and 3D pr…
We develop a method based on the cross-spectrum of an intensity-modulated CW laser, which can extract a signal from an extremely noisy environment and image objects hidden in turbid media. We theoretically analyzed our scheme and performed the experiment by scanning the object placed in between two ground glass diffusers. The image of the object is retrieved by collecting the amplitudes at the modulation frequency of all the cross-spectra. Our method is non-invasive, easy-to-implement, and can work for both static and dynamic media.
On-invasive optical imaging techniques are essential diagnostic tools in many fields. Although various recent methods have been proposed to utilize and control light in multiple scattering media, non-invasive optical imaging through and inside scattering layers across a large field of view remains elusive due to the physical limits set by the optical memory effect, especially without wavefront shaping techniques. Here, we demonstrate an approach that enables non-invasive fluorescence imaging behind scattering layers with field-of-views extending well beyond the optical memory effect. The metho…
We present a method for single-shot three-dimensional imaging through scattering media with a three-dimensional memory effect. In the proposed computational process, a captured speckle image is two-dimensionally correlated with different scales, and the object is three-dimensionally recovered with three-dimensional phase retrieval. Our method was experimentally demonstrated with a lensless setup and was compared with a multi-shot approach used in our previous work [Y. Okamoto, et al., Opt. Lett. 44, 2526-2529 (2019)].
We demonstrate focusing and imaging through a scattering medium noninvasively by using wavefront shaping. Our concept is based on utilizing the spatial fluorescence contrast which naturally exists in the hidden target object. By scanning the angle of incidence of the illuminating laser beam and maximizing the variation of the detected fluorescence signal from the object, as measured by a bucket detector at the front of the scattering medium, we are able to generate a tightly focused excitation spot. Thereafter, an image is obtained by scanning the focus over the object. The requirements for ap…
Extending super-resolution imaging techniques to objects hidden in strongly scattering media potentially revolutionize the technical analysis for much broader categories of samples, such as biological tissues. The main challenge is the media's inhomogeneous structures which scramble the light path and create noise-like speckle patterns, hindering the object's visualization even at a low-resolution level. Here, we propose a computational method relying on the object's spatial and temporal fluctuation to visualize nanoscale objects through scattering media non-invasively. The fluctuating object …
Optical imaging through complex media, such as biological tissues or fog, is challenging due to light scattering. In the multiple scattering regime, wavefront shaping provides an effective method to retrieve information; it relies on measuring how the propagation of different optical wavefronts are impacted by scattering. Based on this principle, several wavefront shaping techniques were successfully developed, but most of them are highly invasive and limited to proof-of-principle experiments. Here, we propose to use a neural network approach to non-invasively characterize and control light sc…
We present a technique for mapping the complete 3D spatial intensity profile of a laser beam from its fluorescence in an atomic vapour. We propagate shaped light through a rubidium vapour cell and record the resonant scattering from the side. From a single measurement we obtain a camera limited resolution of 200 x 200 transverse points and 659 longitudinal points. In constrast to invasive methods in which the camera is placed in the beam path, our method is capable of measuring patterns formed by counterpropagating laser beams. It has high resolution in all 3 dimensions, is fast and can be com…
Several matrix approaches were developed to control light propagation through multiple scattering media under illumination of ultrashort pulses of light. These matrices can be recorded either with spectral or temporal resolution. Thanks to wavefront shaping, temporal and spatial refocusing have been demonstrated. In this work, we study how these different methods can be exploited to enhance a two-photon excitation fluorescence process. We first compare the different techniques on micrometer-size isolated fluorescent beads. We then demonstrate point-scanning imaging of such fluorescent microbea…
Optical imaging through scattering media is a long-standing challenge. Although many approaches have been developed to focus light or image objects through scattering media, they are either invasive, restricted to stationary or slowly-moving media, or require high-resolution cameras and complex algorithms to retrieve the images. Here we introduce a computational imaging technique that can overcome these restrictions by exploiting spatial-temporal encoded patterns (STEP). We present non-invasive imaging through scattering media with a single-pixel photodetector. We show that the method is insen…
We propose a new method to image fluorescent objects through turbid media base on Airy beam scanning. This is achieved by using the non-diffractive nature of Airy beams, namely their ability to maintain their shape while penetrating through a highly scattering medium. We show, that our technique can image fluorescent objects immersed in turbid media with higher resolution and signal to noise than confocal imaging. As proof-of-principle, we demonstrate imaging of 1$μ$m sized fluorescent beads through a dense suspension of yeast cells with an attenuation coefficient of 51cm$^{-1}$ at a depth of …
Super-resolution imaging with advanced optical systems has been revolutionizing technical analysis in various fields from biological to physical sciences. However, many objects are hidden by strongly scattering media such as rough wall corners or biological tissues that scramble light paths, create speckle patterns and hinder object's visualization, let alone super-resolution imaging. Here, we realize a method to do non-invasive super-resolution imaging through scattering media based on stochastic optical scattering localization imaging (SOSLI) technique. Simply by capturing multiple speckle p…
A holistic approach using non-invasive multimodal imaging and spectroscopic techniques to study the materials (pigments, drawing materials and paper) and painting techniques of watercolour paintings is presented. The non-invasive imaging and spectroscopic techniques include VIS-NIR reflectance spectroscopy and multispectral imaging, micro-Raman spectroscopy, X-ray fluorescence spectroscopy (XRF) and optical coherence tomography (OCT). The three spectroscopic techniques complement each other in pigment identification. Multispectral imaging (near infrared bands), OCT and micro-Raman complement e…
When waves propagate through a strongly scattering medium the energy is transferred to the incoherent wave part by scattering. The wave intensity then forms a random speckle pattern seemingly without much useful information. However, a number of recent physical experiments show how one can extract useful information from this speckle pattern. Here we present the mathematical analysis that explains the quite stunning performance of such a scheme for speckle imaging. Our analysis identifies a scaling regime where the scheme works well. This regime is the white-noise paraxial regime, which leads …
EvidenceMoE: A Physics-Guided Mixture-of-Experts with Evidential Critics for Advancing Fluorescence Light Detection and Ranging in Scattering Media
Center for Modeling, Simulation, & Imaging in Medicine（建模、仿真与成像医学中心）；Rensselaer Polytechnic Institute（伦斯勒理工学院）；University at Albany（阿尔巴尼大学）；IBM T.J. Watson Research Center（IBM 沃森研究中心）
Fluorescence microscopy is widely used in biological imaging, however scattering from tissues strongly limits its applicability to a shallow depth. In this work we adapt a methodology inspired from stellar speckle interferometry, and exploit the optical memory effect to enable fluorescence microscopy through a turbid layer. We demonstrate efficient reconstruction of micrometer-size fluorescent objects behind a scattering medium in epi-microscopy, and study the specificities of this imaging modality (magnification, field of view, resolution) as compared to traditional microscopy. Using a modifi…
Laser Pulse Diagnostics of Ultrafast <8 fs Pulses Through Two-Photon Absorption Fluorescence In Liquid Media -- The Role of GVD and Third-Order Dispersion
We present a minimally-invasive endoscope based on a multimode fiber that combines photoacoustic and fluorescence sensing. From the measurement of a transmission matrix during a prior calibration step, a focused spot is produced and raster-scanned over a sample at the distal tip of the fiber by use of a fast spatial light modulator. An ultra-sensitive fiber-optic ultrasound sensor for photoacoustic detection placed next to the fiber is combined with a photodetector to obtain both fluorescence and photoacoustic images with a distal imaging tip no larger than 250um. The high signal-to-noise rati…
Non-degradable fluorophores that accumulate as Lipofuscin in retinal pigment epithelium (RPE) cells has been a major source of intrinsic biomarker for quantifying the progression of several diseases, including age-related macular degeneration, Stargardt disease and, more. Recent progression in quantifying these diseases is entertained mostly by a few noninvasive imaging techniques, relied either on the life-time of the retinoid-fluorophores or, their linear and, non-linear absorption cross-sections. Apart from these intrinsic properties, a native, excited state dipole-dipole interaction mediat…
The efficient optical tool for elimination of the phase and amplitude distortions produced by imperfectness of the optical elements in microscope lightpath considered. This robust procedure described by simple theoretical model proved to be successful to repair the noisy images with noise to signal ratio close to 100. It is shown that Van Cittert - Zernike theorem provides adequate description of imperfect microscope operating under both coherent and incoherent illumination conditions and having turbid media in object and intermediate planes.
The ability to steer light propagation inside scattering media has long been sought-after due to its potential widespread applications. To form optical foci inside scattering media, the only feasible strategy is to guide photons by using either implanted or virtual guide stars. However, all of these guide stars must be introduced extrinsically, either invasively or by physical contact, limiting the scope of their application. Here, we focus light inside scattering media by employing intrinsic dynamics as guide stars. By time-reversing the perturbed component of the scattered light adaptively, …
Scattering can rapidly degrade our ability to form an optical image, to the point where only speckle-like patterns can be measured. Truly non-invasive imaging through a strongly scattering obstacle is difficult, and usually reliant on a computationally intensive numerical reconstruction. In this work we show that, by combining the cross-correlations of the measured speckle pattern at different times, it is possible to track a moving object with minimal computational effort and over a large field of view.
Optical imaging through complex scattering media is one of the major technical challenges with important applications in many research fields, ranging from biomedical imaging, astronomical telescopy, and spatially multiplex optical communications. Although various approaches for imaging though turbid layer have been recently proposed, they had been limited to two-dimensional imaging. Here we propose and experimentally demonstrate an approach for three-dimensional single-shot imaging of objects hidden behind an opaque scattering layer. We demonstrate that under suitable conditions, it is possib…
In turbid media, scattering of light scrambles information of the incident beam and represents an obstacle to optical imaging. Noninvasive imaging through opaque layers is challenging for dynamic and wide-field objects due to unreliable image reconstruction processes. We here propose a new perspective to solve these problems: rather than using the full point-spread-function (PSF), the wave distortions in scattering layers can be characterized with only the phase of the optical-transfer-function (OTF, the Fourier transform of PSF), with which diffraction-limit images can be analytically solved.…
In spite of the crucial role it is believed to play in nature, fluorescence in natural organisms remains under-investigated from optical, chemical and biological perspectives. One example is the transparent wings of insects from the order Hemiptera: fluorescence emission has so far not been reported in any of the 3,000 described species of the superfamily Cicadoidea. The phenomenon has however been investigated in several butterfly and moth species, in which the coloured wing scales are known to embed fluorophores such as papiliochrome II. Photonic structures present in these scales may mediat…
Optical wavefront-shaping has emerged as a powerful tool to manipulate light in strongly scattering media. It enables diffraction-limited focusing and imaging at depths where conventional microscopy techniques fail. However, while most wavefront-shaping works to-date exploited direct access to the target or implanted probes, the challenge is to apply it non-invasively inside complex samples. Ultrasonic-tagging techniques have been recently demonstrated but these require a sequential point-by- point acquisition, a major drawback for imaging applications. Here, we introduce a novel approach to n…
Fluorescence microscopy has revolutionized biomedical research over the past three decades. Its high molecular specificity and unrivaled single molecule level sensitivity have enabled breakthroughs in a variety of research fields. For in vivo applications, its major limitation is the superficial imaging depth as random scattering in biological tissues causes exponential attenuation of the ballistic component of a light wave. Here we present fluorescence microscopy beyond the ballistic regime by combining single cycle pulsed ultrasound modulation and digital optical phase conjugation. We demons…
Focusing light into opaque random or scattering media such as biological tissue is a much sought-after goal for biomedical applications such as photodynamic therapy, optical manipulation, and photostimulation. However, focusing with conventional lenses is restricted to one transport mean free path in scattering media, limiting both optical penetration depth and resolution. Focusing deeper is possible by using optical phase conjugation or wavefront shaping to compensate for the scattering. For practical applications, wavefront shaping offers the advantage of a robust optical system that is less…
Propagation of intense, ultrashort laser pulses through condensed media like crystals of BaF$_2$ and sapphire results in the formation of filaments. We demonstrate that the onset of filamentation may be controlled by rotating the plane of polarization of incident light. We directly visualize filamentation in BaF_2 via six-photon absorption-induced fluorescence and, concomitantly, by probing the spectral and spatial properties of white light that is generated.
The small correction volume for conventional wavefront shaping methods limits their applications in biological imaging through scattering media. We demonstrate large volume wavefront shaping through a scattering layer with a single correction by conjugate adaptive optics and remote focusing (CAORF). The remote focusing module can keep the conjugation between the AO and scattering layer during three-dimensional scanning. This new configuration provides a wider correction volume by the best utilization of the memory effect in a fast three-dimensional laser scanning microscope. Our results show t…
We experimentally demonstrate optical control of filamentation that occurs during propagation of intense, ultrashort laser pulses through crystals like barium fluoride and sapphire. Control is exercised by rotating the plane of polarization of the incident laser radiation and is demonstrated by directly visualizing filamentation in the bulk via six-photon absorption-induced fluorescence and, concomitantly, by probing the spectral and spatial properties of white light that is generated.
Imaging with optical resolution through and inside complex samples is a difficult challenge with important applications in many fields. The fundamental problem is that inhomogeneous samples, such as biological tissues, randomly scatter and diffuse light, impeding conventional image formation. Despite many advancements, no current method enables to noninvasively image in real-time using diffused light. Here, we show that owing to the memory-effect for speckle correlations, a single image of the scattered light, captured with a standard high-resolution camera, encodes all the information that is…
We introduce a generalized version of phase retrieval called multiplexed phase retrieval. We want to recover the phase of amplitude-only measurements from linear combinations of them. This corresponds to the case in which multiple incoherent sources are sampled jointly, and one would like to recover their individual contributions. We show that a recent spectral method developed for phase retrieval can be generalized to this setting, and that its performance follows a phase transition behavior. We apply this new technique to light focusing at depth in a complex medium. Experimentally, although …
Optical time-reversal techniques are being actively developed to focus light through or inside opaque scattering media. When applied to biological tissue, these techniques promise to revolutionize biophotonics by enabling deep-tissue non-invasive optical imaging, optogenetics, optical tweezers and photodynamic therapy. In all previous optical time-reversal experiments, the scattered light field was well-sampled during wavefront measurement and wavefront reconstruction, following the Nyquist sampling criterion. Here, we overturn this conventional practice by demonstrating that even when the sca…
Focusing scattered light using wavefront shaping provides interesting perspectives to image deep in opaque samples, as e.g. in nonlinear fluorescence microscopy. Applying these technics to in vivo imaging remains challenging due to the short decorrelation time of the speckle in depth, as focusing and imaging has to be achieved within the order of the decorrelation time. In this paper, we experimentally study the focus lifetime after focusing through dynamical scattering media, when iterative wavefront optimization and speckle decorrelation occur over the same timescale. We show experimental si…
Fluorescence photoacoustic tomography (fPAT) is a molecular imaging modality that combines photoacoustic tomography (PAT) with fluorescence imaging to obtain high-resolution imaging of fluorescence distributions inside heterogeneous media. The objective of this work is to study inverse problems in the quantitative step of fPAT where we intend to reconstruct physical coefficients in a coupled system of radiative transport equations using internal data recovered from ultrasound measurements. We derive uniqueness and stability results on the inverse problems and develop some efficient algorithms …
Complete polarimeters deliver the full polarization transfer matrix of a medium that relates input polarization states to output polarization states. In order to interpret the Mueller matrix of a luminescent medium at the emission frequency, accountings are required for polarization transformations of the medium at the excitation frequency, the light scattering event, and the polarization transformations at the emission frequency. A general framework for this kind of analysis is presented herein. The fluorescence Mueller matrix is expressed as a product of Mueller matrices of the medium at the…
Light scattering in inhomogeneous media induces wavefront distortions which pose an inherent limitation in many optical applications. Examples range from microscopy and nanosurgery to astronomy. In recent years, ongoing efforts have made the correction of spatial distortions possible by wavefront shaping techniques. However, when ultrashort pulses are employed scattering induces temporal distortions which hinder their use in nonlinear processes such as in multiphoton microscopy and quantum control experiments. Here we show that correction of both spatial and temporal distortions can be attaine…
