Adaptive optics in combination with multi-photon techniques is a powerful approach to image deep into a specimen. Remarkably, virtually all adaptive optics schemes today rely on wavefront modulators which are reflective, diffractive, or both. This, however, can pose a severe limitation for applications. Here, we present a fast and robust sensorless adaptive optics scheme adapted for transmissive wavefront modulators. We study our scheme in numerical simulations and in experiments with a novel, optofluidic wavefront shaping device which is transmissive, refractive, polarisation-independent and broadband. We demonstrate scatter correction of two-photon-excited fluorescence images of microbeads as well as brain cells and benchmark our device against a liquid-crystal spatial light modulator. Our method and technology could open new routes for adaptive optics in scenarios where previously the restriction to reflective and diffractive devices may have staggered innovation and progress.
Adaptive optics (AO) is a powerful image correction technique with proven benefits for many life-science microscopy methods. However, the complexity of adding a reflective wavefront modulator and a wavefront sensor into already complicated microscope has made AO prohibitive for its widespread adaptation in microscopy systems. We present here the design and performance of a compact fluorescence microscope using a fully refractive optofluidic wavefront modulator yielding imaging performance on par with that of conventional deformable mirrors, both in correction fidelity and articulation. We comb…
A novel scheme for tunable optofluidic optical coupler is proposed, by using directional coupling waveguide structure and microfluidic channel with two tapers at end points. The normalized optical power at two output ports can be dynamically manipulated by controlling the refractive index of liquid mixture in microfluidic channel. The optical performance of the designed device is numerically investigated by employing the beam propagation method (BPM). The simulated results demonstrate that large dynamic range and low optical loss for both TE and TM mode can be easily achieved, and furthermore …
We have investigated the bleaching dynamics that occur in optofluidic dye lasers where the liquid laser dye in a microfluidic channel is locally bleached due to optical pumping. We find that for microfluidic devices, the dye bleaching may be compensated through diffusion of dye molecules alone. By relying on diffusion rather than convection to generate the necessary dye replenishment, our observation potentially allows for a significant simplification of optofluidic dye laser device layouts, omitting the need for cumbersome and costly external fluidic handling or on-chip microfluidic pumping d…
We report the implementation of a fully on-chip, lensless microscopy technique termed optofluidic ptychography. This imaging modality complements the miniaturization provided by microfluidics and allows the integration of ptychographic microscopy into various lab-on-a-chip devices. In our prototype, we place a microfluidic channel on the top surface of a coverslip and coat the bottom surface with a scattering layer. The channel and the coated coverslip substrate are then placed on top of an image sensor for diffraction data acquisition. Similar to the operation of flow cytometer, the device ut…
Laser writing of materials is normally performed by the sequential scanning of a single focused beam across a sample. This process is time-consuming and it can severely limit the throughput of laser systems in key applications such as surgery, microelectronics, or manufacturing. Here we report a parallelization strategy based on ultrasound waves in a liquid to diffract light into multiple beamlets. Adjusting amplitude, frequency, or phase of ultrasound allows tunable multi-focus distributions with sub-microsecond control. When combined with sample translation, the dynamic splitting of light le…
As a revolutionary observation tool in life science, biomedical, and material science, optical microscopy allows imaging of samples with high spatial resolution and a wide field of view. However, conventional microscopy methods are limited to single imaging and cannot accomplish real-time image processing. The edge detection, image enhancement and phase visualization schemes have attracted great interest with the rapid development of optical analog computing. The two main physical mechanisms that enable optical analog computing originate from two geometric phases: the spin-redirection Rytov-Vl…
We describe a diffraction microscopy technique based on refractive optics to study structural variations in crystals. The X-ray beam diffracted by a crystal was magnified by beryllium parabolic refractive lenses on a 2D X-ray camera. The microscopy setup was integrated into the 6-circle Huber diffractometer at the ESRF beamline ID06. Our setup allowed us to visualize structural imperfections with a resolution of approximately 1 micrometer. The configuration, however, can easily be adapted for sub-micrometer resolution.
The existing optical microscopes form an image by collecting photons emitted from an object. Here we report on the experimental realization of microscopy without the need for direct optical communication with the sample. To achieve this, we have scanned a single gold nanoparticle acting as a nano-antenna in the near field of a sample and have studied the modification of its intrinsic radiative properties by monitoring its plasmon spectrum.
The application of the non conventional imaging technique LOFI (Laser Optical Feedback Imaging) to coherent microscopy is presented. This simple and efficient technique using frequency-shifted optical feedback needs the sample to be scanned in order to obtain an image. The effects on magnitude and phase signals such as vignetting and field curvature occasioned by the scanning with galvanometric mirrors are discussed. A simple monitoring method based on phase images is proposed to find the optimal position of the scanner. Finally, some experimental results illustrating this technique are presen…
Dual-comb microscopy (DCM), based on a combination of dual-comb spectroscopy (DCS) with two-dimensional spectral encoding (2D-SE), is a promising method for scan-less confocal laser microscopy giving an amplitude and phase image contrast with the confocality. However, signal loss in a 2D-SE optical system hampers increase in image acquisition rate due to decreased signa-to-noise ratio. In this article, we demonstrated optical image amplification in DCM with an erbium-doped fiber amplifier (EDFA). Combined use of image-encoded DCS interferogram and EDFA benefits from not only the batch amplific…
Super-resolution optical microscopy is a rapidly evolving scientific field dedicated to imaging sub-wavelength sized objects, leaving its mark in multiple branches of biology and technology. While several super-resolution optical microscopy methods have become a common tool in life science imaging, new methods, supported by cutting-edge technology, continue to emerge. One rather recent addition to the super-resolution toolbox, image scanning microscopy (ISM), achieves an up to twofold lateral resolution enhancement in a robust and straightforward manner. To further enhance ISM's resolution in …
Adaptive optics is a strategy to compensate for sample-induced aberrations in microscopy applications. Generally, it requires the presence of "guide stars" in the sample to serve as localized reference targets. We describe an implementation of conjugate adaptive optics that is amenable to widefield (i.e. non-scanning) microscopy, and can provide aberration corrections over potentially large fields of view without the use of guide stars. A unique feature of our implementation is that it is based on wavefront sensing with a single-shot partitioned-aperture sensor that provides large dynamic rang…
One of the fundamental limits of classical optical microscopy is the diffraction limit of optical resolution. It results from the finite bandwidth of the optical transfer function (or OTF) of an optical microscope, which restricts the maximum spatial frequencies that are transmitted by a microscope. However, given the frequency support of the OTF, which is fully determined by the used optical hardware, an open and unsolved question is what is the optimal amplitude and phase distribution of spatial frequencies across this support that delivers the "sharpest" possible image. In this paper, we wi…
We theoretically and experimentally demonstrate a multifrequency excitation and detection scheme in apertureless near field optical microscopy, that exceeds current state of the art sensitivity and background suppression. By exciting the AFM tip at its two first flexural modes, and demodulating the detected signal at the harmonics of their sum, we extract a near field signal with a twofold improved sensitivity and deep sub-wavelength resolution, reaching $λ/230$. Furthermore, the method offers rich control over experimental degrees of freedom, expanding the parameter space for achieving comple…
Photoemission electron microscopy was used to image the electrons photoemitted from specially tailored Ag nanoparticles deposited on a Si substrate (with its native oxide SiO$_{x}$). Photoemission was induced by illumination with a Hg UV-lamp (photon energy cutoff $\hbarω_{UV}=5.0$ eV, wavelength $λ_{UV}=250$ nm) and with a Ti:Sapphire femtosecond laser ($\hbarω_{l}=3.1$ eV, $λ_{l}=400$ nm, pulse width below 200 fs), respectively. While homogeneous photoelectron emission from the metal is observed upon illumination at energies above the silver plasmon frequency, at lower photon energies the em…
The imaging performance of an optical microscope can be degraded by sample-induced aberrations. A general strategy to undo the effect of these aberrations is to apply wavefront correction with a deformable mirror (DM). In most cases, the DM is placed conjugate to the microscope pupil, called pupil adaptive optics (AO). When the aberrations are spatially variant, an alternative configuration involves placing the DM conjugate to the main source of aberrations, called conjugate AO. We provide theoretical and experimental comparison of both configurations for the simplified case where spatially va…
We introduce a point-like scanning single-photon source that operates at room temperature and offers an exceptional photostability (no blinking, no bleaching). This is obtained by grafting in a controlled way a diamond nanocrystal (size around 20 nm) with single nitrogen-vacancy color-center occupancy at the apex of an optical probe. As an application, we image metallic nanostructures in the near-field, thereby achieving a near-field scanning single-photon microscopy working at room temperature on the long term. Our work may be of importance to various emerging fields of nanoscience where an a…
We demonstrate a new 'microsphere nanoscope' that uses ordinary SiO2 microspheres as superlenses to create a virtual image of the object in near field. The magnified virtual image greatly overcomes the diffraction limit. We are able to resolve clearly 50-nm objects under a standard white light source in both transmission and reflection modes. The resolution achieved for white light opens a new opportunity to image viruses, DNA and molecules in real time.
Confocal imaging and phase imaging are powerful tools in life science research and industrial inspection. To coherently link the two techniques with different depth resolutions, we introduce an optical frequency comb (OFC) to microscopy. Two-dimensional (2D) image pixels of a sample were encoded onto OFC modes via 2D spectral encoding, in which OFC acted as an optical carrier with a vast number of discrete frequency channels. Then, a scan-less full-field confocal image with a depth resolution of 62.4 um was decoded from a mode-resolved OFC amplitude spectrum obtained by dual-comb spectroscopy.…
Zernike polynomials are widely used mathematical models of experimentally observed optical aberrations. Their useful mathematical properties, in particular their orthogonality, make them a ubiquitous basis set for solving various problems in beam optics. Thus they have found widespread use in adaptive optics realizations that are used to correct wavefront aberrations. However, Zernike aberrations lose their orthogonality when used in combination with Gaussian beams, which are omnipresent in real-world optical applications. As a consequence, Zernike aberrations in Gaussian beams start to cross-…
Magnetic and spintronic media have offered fundamental scientific subjects and technological applications. Magneto-optic Kerr effect (MOKE) microscopy provides the most accessible platform to study the dynamics of spins, magnetic quasi-particles, and domain walls. However, in the research of nanoscale spin textures and state-of-the-art spintronic devices, optical techniques are generally restricted by the extremely weak magneto-optical activity and diffraction limit. Highly sophisticated, expensive electron microscopy and scanning probe methods thus have come to the forefront. Here, we show th…
An integrated microscope that combines different optical techniques for simultaneous imaging is demonstrated. The microscope enables spectral-domain optical coherence microscopy based on optical backscatter, and multi-photon microscopy for the detection of two-photon fluorescence and second harmonic generation signals. The unique configuration of this integrated microscope allows for the simultaneous acquisition of both anatomical (structural) and functional imaging information with particular emphasis for applications in the fields of tissue engineering and cell biology. In addition, the cont…
Sample-induced image-degradation remains an intricate wave-optical problem in light-sheet microscopy. Here we present biobeam, an open-source software package that enables to simulate operational light-sheet microscopes by combining data from 10^5 - 10^6 multiplexed and GPU-accelerated point-spread-function calculations. The wave-optical nature of these simulations leads to the faithful reproduction of spatially varying aberrations, diffraction artifacts, geometric image distortions, adaptive optics, and emergent wave optical phenomena, and renders image-formation in light-sheet microscopy com…
We demonstrate a new versatile method for 3D super-resolution microscopy by using a deformable mirror to shape the point spread function of our microscope in a continuous and controllable way. We apply this for 3D STORM imaging of microtubules.
Center of Advanced European Studies and Research (caesar)（欧洲高级研究中心 (caesar)）
Aberrations limit scanning fluorescence microscopy when imaging in scattering materials such as biological tissue. Model-based approaches for adaptive optics take advantage of a computational model of the optical setup. Such models can be combined with the optimization techniques of machine learning frameworks to find aberration corrections, as was demonstrated for focusing a laser beam through aberrations onto a camera [arXiv:2007.13400]. Here, we extend this approach to two-photon scanning microscopy. The developed sensorless technique finds corrections for aberrations in scattering samples …
Scattering scanning near-field optical microscopy enables optical imaging and characterization of plasmonic devices with nanometer-scale resolution well below the diffraction limit. This technique enables developers to probe and understand the waveguide-coupled plasmonic antenna in as-fabricated heat-assisted magnetic recording heads. In order validate and predict results and to extract information from experimental measurements that is physically comparable to simulations, a model was developed to translate the simulated electric field into expected near-field measurements using physical para…
Super-resolution fluorescence microscopy is an important tool in biomedical research for its ability to discern features smaller than the diffraction limit. However, due to its difficult implementation and high cost, the universal application of super-resolution microscopy is not feasible. In this paper, we propose and demonstrate a new kind of super-resolution fluorescence microscopy that can be easily implemented and requires neither additional hardware nor complex post-processing. The microscopy is based on the principle of stepwise optical saturation (SOS), where $M$ steps of raw fluoresce…
We present a method to realize active optical tips for use in near-field optics that can operate at room temperature. A metal-coated optical tip is covered with a thin polymer layer stained with CdSe nanocrystals or nanorods at low density. The time analysis of the emission rate and emission spectra of the active tips reveal that a very small number of particles - possibly down to only one - can be made active at the tip apex. This opens the way to near-field optics with a single inorganic nanoparticle as a light source.
As an important branch of photoacoustic microscopy, optical-resolution photoacoustic microscopy suffers from limited depth of field due to the strongly focused laser beam. In this work, a 3D information fusion algorithm based on 3D stationary wavelet transform and joint weighted evaluation optimization is proposed to fuse multi-focus photoacoustic data to achieve large-volumetric and high-resolution 3D imaging. First, a three-dimensional stationary wavelet transform was performed on the multi-focus data to obtain eight wavelet coefficients. Differential evolution algorithm based on joint weigh…
Accelerating imaging speed in optical microscopy is often realized at the expense of image contrast, image resolution, and detection sensitivity- a common predicament for advancing high-speed and high-throughput cellular imaging. We here demonstrate a new imaging approach, called asymmetric-detection time-stretch optical microscopy (ATOM), which can deliver ultrafast label-free high-contrast flow imaging with well delineated cellular morphological resolution and in-line optical image amplification to overcome the compromised imaging sensitivity at high speed. We show that ATOM can separately r…
University of California, Los Angeles（加州大学洛杉矶分校）；California NanoSystems Institute（加州纳米系统研究所）
We discuss recently emerging applications of the state-of-art deep learning methods on optical microscopy and microscopic image reconstruction, which enable new transformations among different modes and modalities of microscopic imaging, driven entirely by image data. We believe that deep learning will fundamentally change both the hardware and image reconstruction methods used in optical microscopy in a holistic manner.
We demonstrate orientation-sensitive multimodal nonlinear optical polarizing microscopy capable of probing orientational, polar, and biaxial features of mesomorphic ordering in soft matter. This technique achieves simultaneous imaging in broadband coherent anti-Stokes Raman scattering, multi-photon excitation fluorescence, and multi-harmonic generation polarizing microscopy modes and is based on the use of a single femtosecond laser and a photonic crystal fiber as sources of the probing light. We show the viability of this technique for mapping of three dimensional patterns of molecular orient…
We demonstrate an electro-optic wide-field method to enable fluorescence lifetime microscopy (FLIM) with high throughput and single-molecule sensitivity. Resonantly driven Pockels cells are used to efficiently gate images at 39 MHz, allowing fluorescence lifetime to be captured on standard camera sensors. Lifetime imaging of single molecules is enabled in wide-field with exposure times of less than 100 milliseconds. This capability allows combination of wide-field FLIM with single-molecule super-resolution localization microscopy. Fast single-molecule dynamics such as FRET and molecular bindin…
We present a simple and compact approach of structured illumination microscopy by using three $2\times2$ fiber couplers and one $1\times4$ MEMS optics switch. One uniform and three fringe illumination patterns were produced by placing seven output fiber tips at the conjugate Fourier plane of the illumination path. Stable and relatively high-speed illumination switching was achieved by the optics switch. Super-resolution and optical sectioned information was reconstructed from 4-frame data by using algorithms based on a joint Richardson-Lucy deconvolution method and a Hilbert transform method. …
Multifocal plane microscopy (MUM) allows three dimensional objects to be imaged in a single camera frame. Our approach uses dual orthogonal diffraction phase gratings with a quadratic distortion of the lines to apply defocus to the first diffraction orders which, when paired with a relay lens, allows for 9 focal planes to be imaged on a single camera chip. This approach requires a strong signal level to ensure sufficient intensity in the diffracted light, but has the advantage of being compact and straightforward to implement. As the microscope begins to focus deeper into the sample, aberratio…
Halide perovskites have remarkable properties for relatively crudely processed semiconductors, including large optical absorption coefficients and long charge carrier lifetimes. Thanks to such properties, these materials are now competing with established technologies for use in cost-effective and efficient light harvesting and light emitting devices. Nevertheless, our fundamental understanding of the behaviour of charge carriers in these materials particularly on the nano to micro scale has on the whole lagged behind the empirical device performances. Such understanding is essential to contro…
To enhance the resolution of a confocal laser scanning microscope the additional information of a pinhole plane image taken at every excitation scan position can be used [C. J. R. Sheppard, Super-resolution in confocal imaging, Optik 80, 5354 (1988)]. This photon reassignment principle is based on the fact that the most probable position of an emitter is at half way between the nominal focus of the excitation laser and the position corresponding to the (off centre) detection position. Therefore, by reassigning the detected photons to this place, an image with enhanced detection efficiency and …
