Photoluminesce (PL) spectroscopy offers excellent methods for mapping the PL decay on the nanosecond time scale. However, capturing maps of emission dynamics on the microsecond time scale can be highly time-consuming. We present a new approach to fluorescence lifetime imaging FLIM, which combines the concept of random temporal speckles excitation (RATS) with the concept of a single-pixel camera based on spatial speckles. The spatio-temporal speckle pattern makes it possible to map PL dynamics with unmatched simplicity. Moreover, the method can acquire all data necessary to map PL decays in the microsecond timescale within minutes. We present proof of principle measurements for two samples and compare the reconstructed decays to the non-imaging measurements. Finally, we discuss the effect of the preprocessing routine and other factors on the reconstruction noise level. The presented method is suitable for lifetime imaging processes in several samples, including charge carrier dynamics in perovskites or monitoring solid-state luminophores with a long lifetime of PL.
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…
Nonlinear structured illumination microscopy (nSIM) is an effective approach for super-resolution wide-field fluorescence microscopy with a theoretically unlimited resolution. In nSIM, carefully designed, highly-contrasted illumination patterns are combined with the saturation of an optical transition to enable sub-diffraction imaging. While the technique proved useful for two-dimensional imaging, extending it to three-dimensions (3D) is challenging due to the fading/fatigue of organic fluorophores under intense cycling conditions. Here, we present a compressed sensing approach that allows for…
We present an ultra-thin hybrid imaging system based on an optical multimode fiber (MMF) and an optical fiber hydrophone that combines optical resolution photoacoustic and fluorescence microscopy. To control the illumination at the distal tip of the MMF, a digital micromirror device modulates the amplitude of the optical wavefront which is coupled into the MMF. A set of pre-calibrated speckle illuminations combined with a reconstruction algorithm enables photoacoustic and fluorescence imaging of samples located at the distal tip of the fiber with optical resolution determined by the numerical …
Fluorescence microscopy is indispensable in nanoscience and biological sciences. The versatility of labeling target structures with fluorescent dyes permits to visualize structure and function at a subcellular resolution with a wide field of view. Due to the diffraction limit, conventional optical microscopes are limited to resolving structures larger than 200 nm. The resolution can be enhanced by near-field and far-field super-resolution microscopy methods. Near-field methods typically have a limited field of view and far-field methods are limited by the involved conventional optics. Here, we…
Speckle patterns have been widely used in imaging techniques such as ghost imaging, dynamic speckle illumination microscopy, structured illumination microscopy, and photoacoustic fluctuation imaging. Recent advances in the ability to control the statistical properties of speckles has enabled the customization of speckle patterns for specific imaging applications. In this work, we design and create special speckle patterns for parallelized nonlinear pattern-illumination microscopy based on fluorescence photoswitching. We present a proof-of-principle experimental demonstration where we obtain a …
Beam self-cleaning (BSC) in graded-index (GRIN) multimode fibres (MMFs) has been recently reported by different research groups. Driven by the interplay between Kerr effect and beam self-imaging, BSC counteracts random mode coupling, and forces laser beams to recover a quasi-single mode profile at the output of GRIN fibres. Here we show that the associated self-induced spatiotemporal reshaping allows for improving the performances of nonlinear fluorescence microscopy and endoscopy using multimode optical fibres. We experimentally demonstrate that the beam brightness increase, induced by self-c…
The multiple scattering of coherent light is a problem of both fundamental and applied importance. In optics, phase conjugation allows spatial focussing and imaging through a multiply scattering medium; however, temporal control is nonetheless elusive, and multiple scattering remains a challenge for femtosecond science. Here, we report on the spatially and temporally resolved measurement of a speckle field produced by the propagation of an ultrafast optical pulse through a thick strongly scattering medium. Using spectral pulse shaping, we demonstrate the spatially localized temporal recompress…
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…
We discuss the far-field spatio-temporal cross-correlations of waves multiple-scattered in a turbid medium in which is embedded a hidden heterogeneous region (inclusion) characterized by a distinct scatterer dynamics (as compared to the rest of the medium). We show that the spatio-temporal correlation is affected by the inclusion which suggests a new method of imaging in turbid media. Our results allow qualitative interpretation in terms of diffraction theory: the cross-correlation of scattered waves behaves similarly to the intensity of a wave diffracted by an aperture.
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…
Spatial coherence quantifies spatial field correlations over time, and is one of the fundamental properties of light. Here we investigate the spatial coherence of highly multimode lasers in the regime of short time scales. Counter intuitively, we show that in this regime, the temporal (longitudinal) modes play a crucial role in spatial coherence reduction. To evaluate the spatial coherence we measured the temporal dynamics of speckle fields generated by a highly multimode laser with over $10^5$ lasing spatial (transverse) modes, and examined the dependence of speckle contrast on the exposure t…
We introduce the basic elements of a spatio-angular theory of fluorescence microscopy, providing a unified framework for analyzing systems that image single fluorescent dipoles and ensembles of overlapping dipoles that label biological molecules. We model an aplanatic microscope imaging an ensemble of in-focus fluorescent dipoles as a linear Hilbert-space operator, and we show that the operator takes a particularly convenient form when expressed in a basis of complex exponentials and spherical harmonics$-$a form we call the dipole spatio-angular transfer function. We discuss the implications o…
Mid-infrared photothermal microscopy is a new chemical imaging technology in which a visible beam senses the photothermal effect induced by a pulsed infrared laser. This technology provides infrared spectroscopic information at sub-micron spatial resolution and enables infrared spectroscopy and imaging of living cells and organisms. Yet, current mid-infrared photothermal imaging sensitivity suffers from a weak dependance of scattering on temperature and the image quality is vulnerable to the speckles caused by scattering. Here, we present a novel version of mid-infrared photothermal microscopy…
We investigate the properties of a single-view fluorescence microscope in a 4$f$ geometry when imaging fluorescent dipoles without using the monopole or scalar approximations. We show that this imaging system has a spatio-angular band limit, and we exploit the band limit to perform efficient simulations. Notably, we show that information about the out-of-plane orientation of ensembles of in-focus fluorophores is recorded by paraxial fluorescence microscopes. Additionally, we show that the monopole approximation may cause biased estimates of fluorophore concentrations, but these biases are smal…
Self-Portrait of the Focusing Process in Speckle: III. Tailoring Complex Spatio-Temporal Focusing Laws To Overcome Reverberations in Reflection Imaging
High-content biological microscopy targets high-resolution imaging across large fields-of-view (FOVs). Recent works have demonstrated that computational imaging can provide efficient solutions for high-content microscopy. Here, we use speckle structured illumination microscopy (SIM) as a robust and cost-effective solution for high-content fluorescence microscopy with simultaneous high-content quantitative phase (QP). This multi-modal compatibility is essential for studies requiring cross-correlative biological analysis. Our method uses laterally-translated Scotch tape to generate high-resoluti…
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…
Encoding of spectral information onto monochrome imaging cameras is of interest for wavelength multiplexing and hyperspectral imaging applications. Here, the complex spatio-spectral response of a disordered material is used to demonstrate retrieval of a number of discrete wavelengths over a wide spectral range. Strong, diffuse light scattering in a semiconductor nanowire mat is used to achieve a highly compact spectrometer of micrometer thickness, transforming different wavelengths into distinct speckle patterns with nanometer sensitivity. Spatial multiplexing is achieved through the use of a …
We report sensing of single nanoparticles using disordered metallic nanoisland substrates supporting surface plasmon polaritons (SPPs). Speckle patterns arising from leakage radiation of elastically scattered SPPs provides a unique fingerprint of the scattering microstructure at the sensor surface. Experimental measurements of the speckle decorrelation are presented and shown to enable detection of sorption of individual gold nanoparticles and polystyrene beads. Our approach is verified through bright-field and fluorescence imaging of particles adhering to the nanoisland substrate.
Forward-viewing endoscopic optical coherence tomography (OCT) provides 3D imaging in vivo, and can be combined with widefield fluorescence imaging by use of a double-clad fiber. However, it is technically challenging to build a high-performance miniaturized 2D scanning system with a large field-of-view. In this paper we demonstrate how a 1D scanning probe, which produces cross-sectional OCT images (B-scans) and 1D fluorescence T-scans, can be transformed into a 2D scanning probe by manual scanning along the second axis. OCT volumes are assembled from the B-scans using speckle decorrelation mea…
Long-range speckle correlations play an essential role in wave transport through disordered media, but have rarely been studied in other complex systems. Here we discover spatio-temporal intensity correlations for an optical pulse propagating through a multimode fiber with strong random mode coupling. Positive long-range correlations arise from multiple scattering in fiber mode space and depend on the statistical distribution of arrival times. By optimizing the incident wavefront of a pulse, we maximize the power transmitted at a selected time, and such control is significantly enhanced by the…
Multimode fibers are widely explored for optical communication, imaging and sensing applications. The interference of fiber guided modes generates a speckle pattern, which has been used for high-precision spectroscopy, temperature, and strain sensing. Here we demonstrate a single-shot full-field temporal measurement technique based on a multimode fiber. The complex spatiotemporal speckle field is created by a reference pulse propagating through the fiber, and it interferes with a signal pulse. From the time-integrated interference pattern, both the amplitude and the phase of the signal are ret…
One of the greatest needs in modern ophthalmology is access to the eye choroid in vivo. Without it, it is difficult to introduce new therapies and to understand most of the pathological changes in the eye, because of the total blood flow in the eye is caused by the choroidal circulation. It is entirely responsible for the metabolism of photoreceptors, which each of us feels every day and senses when its deficits begin. Optical selection of choroid and choriocapillaris by currently used imaging techniques such as indocyanine angiography, scanning laser ophthalmoscopy or optical coherence tomogr…
The scattering of multispectral incoherent light is a common and unfavorable signal scrambling in natural scenes. However, the blurred light spot due to scattering still holds lots of information remaining to be explored. Former methods failed to recover the polarized hyperspectral information from scattered incoherent light or relied on additional dispersion elements. Here we put forward the transmission matrix (TM) approach for extended objects under incoherent illumination by speculating the unknown TM through experimentally calibrated or digitally emulated ways. Employing a fiber bundle as…
By circumventing the optical diffraction limit, super-resolved fluorescence microscopies enable the study of larger cellular structures and molecular assemblies. However, fluorescence nanoscopy currently lacks the spatiotemporal resolution to resolve distances on the size of individual molecules and reveal the conformational fine structure and dynamics of molecular complexes. Here we establish FRET nanoscopy by combining colocalization STED microscopy with multiparameter FRET spectroscopy. We simultaneously localize donor and acceptor dyes of single FRET pairs with nanometer resolution and qua…
Fluorescence polarization microscopy images both the intensity and orientation of fluorescent dipoles, which plays a vital role in studying the molecular structure and dynamics of bio-complex. However, it is difficult to resolve the dipole assemblies on the subcellular structure and their dynamics in living cells with super-resolution. Here we report polarized structured illumination microscopy (pSIM), which decouples the entangled spatial and angular structured illumination through interpreting the dipoles in spatio-angular hyperspace. We demonstrate its application on a series of biological …
Duke University（杜克大学）；Tsinghua University（清华大学）；Lund University（隆德大学）
Fast noninvasive probing of spatially varying decorrelating events, such as cerebral blood flow beneath the human skull, is an essential task in various scientific and clinical settings. One of the primary optical techniques used is diffuse correlation spectroscopy (DCS), whose classical implementation uses a single or few single-photon detectors, resulting in poor spatial localization accuracy and relatively low temporal resolution. Here, we propose a technique termed Classifying Rapid decorrelation Events via Parallelized single photon dEtection (CREPE)}, a new form of DCS that can probe and…
