High space-bandwidth product with high spatial phase sensitivity is indispensable for a single-shot quantitative phase microscopy (QPM) system. It opens avenue for widespread applications of QPM in the field of biomedical imaging. Temporally low coherence length light sources are generally implemented to achieve high spatial phase sensitivity in QPM at the cost of either reduced temporal resolution or smaller field of view (FOV). On the contrary, high temporal coherence light sources like lasers are capable of exploiting the full FOV of the QPM systems at the expense of less spatial phase sensitivity. In the present work, we employed pseudo-thermal light source (PTLS) in QPM which overcomes the limitations of conventional light sources. The capabilities of PTLS over conventional light sources are systematically studied and demonstrated on various test objects like USAF resolution chart and thin optical waveguide (height ~ 8 nm). The spatial phase sensitivity of QPM in case of PTLS is measured to be equivalent to that for white light source. The high-speed and large FOV capabilities of PTLS based QPM is demonstrated by high-speed imaging of live sperm cells that is limited by the camera speed and by imaging extra-ordinary large FOV phase imaging on histopathology placenta tissue samples.
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…
We report a novel extension to structured illumination (SI) microscopy that utilizes the Talbot self-imaging effect to generate a SI pattern on a sample with field-of-view (FOV) and resolution unconstrained by the numerical aperture (NA) of the microscope objective. This enables high-resolution SI pattern generation (up to the free-space propagation limit) across large FOVs, which in turn allows SI microscopy to be efficiently adapted for high-throughput imaging. We demonstrated this concept using a simple prototype microscope to image a large FOV (450 x 350 um) with a diffraction-limited fluo…
We present a theoretical investigation of coherent anti-Stokes Raman scattering (CARS) that is modulated by periodically depleting the ground state population through Rabi oscillations driven by an additional control laser. We find that such a process generates optical sidebands in the CARS spectrum and that the frequency of the sidebands depends on the intensity of the control laser light field. We show that analyzing the sideband frequency upon scanning the beams across the sample allows one to spatially resolve emitter positions where a spatial resolution of 65 nm, which is well below the d…
Mid-infrared photothermal (MIP) microscopy has been a promising label-free chemical imaging technique for functional characterization of specimens owing to its enhanced spatial resolution and high specificity. Recently developed wide-field MIP imaging modalities have drastically improved speed and enabled high-throughput imaging of micron-scale subjects. However, the weakly scattered signal from sub-wavelength particles becomes indistinguishable from the shot-noise as a consequence of the strong background light, leading to limited sensitivity. Here, we demonstrate background-suppressed chemic…
Quantitative phase imaging (QPI) is a valuable label-free modality that has gained significant interest due to its wide potentials, from basic biology to clinical applications. Most existing QPI systems measure microscopic objects via interferometry or nonlinear iterative phase reconstructions from intensity measurements. However, all imaging systems compromise spatial resolution for field of view and vice versa, i.e., suffer from a limited space bandwidth product. Current solutions to this problem involve computational phase retrieval algorithms, which are time-consuming and often suffer from…
Whole slide imaging (WSI) has moved digital pathology closer to diagnostic practice in recent years. Due to the inherent tissue topography variability, accurate autofocusing remains a critical challenge for WSI and automated microscopy systems. The traditional focus map surveying method is limited in its ability to acquire a high degree of focus points while still maintaining high throughput. Real-time approaches decouple image acquisition from focusing, thus allowing for rapid scanning while maintaining continuous accurate focus. This work reviews the traditional focus map approach and discus…
Illumination uniformity is a critical parameter for excitation and data extraction quality in widefield biological imaging applications. However, typical imaging systems suffer from spatial and spectral non-uniformity due to non-ideal optical elements, thus require complex solutions for illumination corrections. We present Effective Uniform Color-Light Integration Device (EUCLID), a simple and cost-effective illumination source for uniformity corrections. EUCLID employs a diffuse-reflective, adjustable hollow cavity that allows for uniform mixing of light from discrete light sources and modifi…
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…
Huazhong University of Science and Technology（华中科技大学）；Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics（武汉光电国家研究中心Britton Chance生物医学光子学研究中心）；Massachusetts Institute of Technology（麻省理工学院）；Shenzhen Huazhong University of Science and Technology Research Institute（深圳华中科技大学研究院）
We combine generative adversarial network (GAN) with light microscopy to achieve deep learning super-resolution under a large field of view (FOV). By appropriately adopting prior microscopy data in an adversarial training, the neural network can recover a high-resolution, accurate image of new specimen from its single low-resolution measurement. Its capacity has been broadly demonstrated via imaging various types of samples, such as USAF resolution target, human pathological slides, fluorescence-labelled fibroblast cells, and deep tissues in transgenic mouse brain, by both wide-field and light…
We report a computational 3D microscopy technique, termed Fourier ptychographic diffraction tomography (FPDT), that iteratively stitches together numerous variably illuminated, low-resolution images acquired with a low-numerical aperture (NA) objective in 3D Fourier space to create a wide field-of-view (FOV), high-resolution, depth-resolved complex refractive index (RI) image across large volumes. Unlike conventional optical diffraction tomography (ODT) approaches that rely on controlled bright-field illumination, holographic phase measurement, and high-NA objective detection, FPDT employs tom…
The resonant buildup of light within optical microcavities elevates the radiation pressure which mediates coupling of optical modes to the mechanical modes of a microcavity. Above a certain threshold pump power, regenerative mechanical oscillation occurs causing oscillation of certain mechanical eigenmodes. Here, we present a methodology to spatially image the micro-mechanical resonances of a toroid microcavity using a scanning probe technique. The method relies on recording the induced frequency shift of the mechanical eigenmode when in contact with a scanning probe tip. The method is passive…
The trade-off between throughput and image quality is an inherent challenge in microscopy. To improve throughput, compressive imaging under-samples image signals; the images are then computationally reconstructed by solving a regularized inverse problem. Compared to traditional regularizers, Deep Learning based methods have achieved greater success in compression and image quality. However, the information loss in the acquisition process sets the compression bounds. Further improvement in compression, without compromising the reconstruction quality is thus a challenge. In this work, we propose…
The emergence of far-field super-resolution microscopy has rejuvenated the possibility for nanoscale imaging. Approaches to far-field super-resolution that utilize point scanning often depends on spatially reducing the size of the focused spot. However, the focused spot always achieves high resolution at the expense of extremely low light efficiency for the probing mainlobe and high-intensity sidelobes, which limits the applications in nanoscale imaging and might cause misinterpretation of samples. Here we report a sharp probing spot with the diffraction efficiency of 3.76% at the resolution o…
Increasing demand for understanding the vast heterogeneity of cellular phenotypes has driven the development of imaging flow cytometry (IFC), that combines features of flow cytometry with fluorescence and bright field microscopy. IFC combines the throughput and statistical advantage of flow cytometry with the ability to discretely measure events based on a real or computational image, as well as conventional flow cytometry metrics. A limitation of existing IFC systems is that, regardless of detection methodology, only two-dimensional (2D) cell images are obtained. Without tomographic three-dim…
We demonstrate a motion-free intensity diffraction tomography technique that enables direct inversion of 3D phase and absorption from intensity-only measurements for weakly scattering samples. We derive a novel linear forward model, featuring slice-wise phase and absorption transfer functions using angled illumination. This new framework facilitates flexible and efficient data acquisition, enabling arbitrary sampling of the illumination angles. The reconstruction algorithm performs 3D synthetic aperture using a robust, computation and memory efficient slice-wise deconvolution to achieve resolu…
High Sensitivity Transient Holographic Full-Polarization Microscopy
Institute for Physical Chemistry, Ruprecht-Karls-University Heidelberg（海德堡大学物理化学研究所）；Ruprecht-Karls-University Heidelberg（海德堡大学）；Karlsruhe Institute of Technology（卡尔斯鲁厄理工学院）
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…
In this article, we report an imaging method, termed Fourier ptychographic microscopy (FPM), which iteratively stitches together a number of variably illuminated, low-resolution intensity images in Fourier space to produce a wide-field, high-resolution complex sample image. By adopting a wavefront correction strategy, the FPM method can also correct for aberrations and digitally extend a microscope's depth-of-focus beyond the physical limitations of its optics. As a demonstration, we built a microscope prototype with a resolution of 0.78 um, a field-of-view of ~120 mm2, and a resolution-invari…
Fluorescence imaging is the most widely used method for unveiling the molecular composition of biological specimens. However, the weak optical emission of fluorescent probes and the tradeoff between imaging speed and sensitivity is problematic for acquiring blur-free images of fast phenomena, such as sub-millisecond biochemical dynamics in live cells and tissues, and cells flowing at high speed. We report a solution that achieves real-time pixel readout rates one order of magnitude faster than a modern electron multiplier charge coupled device (EMCCD) - the gold standard in high-speed fluoresc…
Structured illumination microscopy (SIM) can double the resolution beyond the light diffraction limit, but it comes at the cost of multiple camera exposures and the heavy computation burden of multiple Fourier transforms. In this paper, we report a novel technique termed single-shot SIM, to overcome these limitations. A multi-task joint deep-learning strategy is proposed. Generative adversative networks (GAN) are employed to generate five structured illumination images based on the single-shot structured illumination image. U-Net is employed to reconstruct the super-resolution image from these…
We theorize the surface step characterization by reflected incoherent-light differential interference microscopy with consideration of the optical diffraction effect. With the integration of localization analysis, we develop a quantitative differential interference optical system, by which we demonstrate that the axial resolution of measuring surface height variation is sensitive to the shear distance between the two spatially differentiated beams. We fabricate three nanometer-size steps by photolithography, and successfully characterize their 1D height variations with 0.13 nm Hz^(-1/2) axial …
Two-Photon Microscopy has become an invaluable tool for biological and medical research, providing high sensitivity, molecular specificity, inherent three-dimensional sub-cellular resolution and deep tissue penetration. In terms of imaging speeds, however, mechanical scanners still limit the acquisition rates to typically 10-100 frames per second. Here we present a high-speed non-linear microscope achieving kilohertz frame rates by employing pulse-modulated, rapidly wavelength-swept lasers and inertia-free beam steering through angular dispersion. In combination with a high bandwidth, single-p…
Wavefront shaping has revolutionized imaging deep in scattering media, being able to spatially and temporally refocus light through or inside the medium. However, wavefront shaping is not compatible yet with polarization-resolved microscopy given the need of polarizing optics to refocus light with a controlled polarization state. Here, we show that wavefront shaping is not only able to restore a focus, but it can also recover the injected polarization state without using any polarizing optics at the detection. This counter-intuitive effect occurs up to several transport mean free path thick sa…
University of California, Los Angeles（加州大学洛杉矶分校）；David Geffen School of Medicine, University of California, Los Angeles（加州大学洛杉矶分校大卫·格芬医学院）
We report a framework based on a generative adversarial network (GAN) that performs high-fidelity color image reconstruction using a single hologram of a sample that is illuminated simultaneously by light at three different wavelengths. The trained network learns to eliminate missing-phase-related artifacts, and generates an accurate color transformation for the reconstructed image. Our framework is experimentally demonstrated using lung and prostate tissue sections that are labeled with different histological stains. This framework is envisaged to be applicable to point-of-care histopathology…
Optical phase-space functions describe spatial and angular information simultaneously; examples include light fields in ray optics and Wigner functions in wave optics. Measurement of phase-space enables digital refocusing, aberration removal and 3D reconstruction. High-resolution capture of 4D phase-space datasets is, however, challenging. Previous scanning approaches are slow, light inefficient and do not achieve diffraction-limited resolution. Here, we propose a multiplexed method that solves these problems. We use a spatial light modulator (SLM) in the pupil plane of a microscope in order t…
Adapting optical microscopy methods for nanoscale characterization of defects in two-dimensional (2D) materials is a vital step for photonic on-chip devices. To increase the analysis throughput, waveguide-based on-chip imaging platforms have been recently developed. Their inherent disadvantage, however, is the necessity to transfer the 2D material from the growth substrate to the imaging chip which introduces contamination, potentially altering the characterization results. Here we present a unique approach to circumvent these shortfalls by directly growing a widely-used 2D material (hexagonal…
Spectral DiffuserScope: a compact snapshot hyperspectral microscope
University of California, Berkeley（加州大学伯克利分校）；UCB/UCSF Joint Graduate Program in Bioengineering, University of California, Berkeley（加州大学伯克利分校生物工程专业联合研究生项目）；Seoul National University（首尔大学）；Kyung Hee University（庆熙大学）；Stanford University（斯坦福大学）；VIAVI Solutions Inc（维亚维解决方案公司）；Cornell University（康奈尔大学）
Spatiotemporal dynamics of excitons in isolated semiconducting single-walled carbon nanotubes are studied using transient absorption microscopy. Differential reflection and transmission of an 810-nm probe pulse after excitation by a 750-nm pump pulse are measured. We observe a bi-exponentially decaying signal with a fast time constant of 0.66 ps and a slower time constant of 2.8 ps. Both constants are independent of the pump fluence. By spatially and temporally resolving the differential reflection, we are able to observe a diffusion of excitons, and measure a diffusion coefficient of 200 cm2/…
Imaging dynamical processes at interfaces and on the nanoscale is of great importance throughout science and technology. While light-optical imaging techniques often cannot provide the necessary spatial resolution, electron-optical techniques damage the specimen and cause dose-induced artefacts. Here, Optical Near-field Electron Microscopy (ONEM) is proposed, an imaging technique that combines non-invasive probing with light, with a high spatial resolution read-out via electron optics. Close to the specimen, the optical near-fields are converted into a spatially varying electron flux using a p…
Automated label-free quantitative imaging of biological samples can greatly benefit high throughput diseases diagnosis. Digital holographic microscopy (DHM) is a powerful quantitative label-free imaging tool that retrieves structural details of cellular samples non-invasively. In off-axis DHM, a proper spatial filtering window in Fourier space is crucial to the quality of reconstructed phase image. Here we describe a region-recognition approach that combines shape recognition with an iterative thresholding to extracts the optimal shape of frequency components. The region recognition technique …
We experimentally demonstrate long-wave infrared-visible sum-frequency generation microscopy for imaging polaritonic resonances of infrared (IR) nanophotonic structures. This nonlinear-optical approach provides direct access to the resonant field enhancement of the polaritonic near fields, while the spatial resolution is limited by the wavelength of the visible sum-frequency signal. As a proof-of-concept, we here study periodic arrays of subdiffractional nanostructures made of 4H-silicon carbide supporting localized surface phonon polaritons. By spatially scanning tightly focused incident beam…
We present a simple and effective instrument for simultaneous real-time imaging and hysteresis of the anisotropic magnetic domain dynamics in thin films using the Magneto-Optical Kerr Effect (MOKE). We furthermore illustrate that magnetic imaging allows a more accurate interpretation of the magnetization reversal processes than the conventional hysteresis characterization. In particular, we present a case where the onset of a double-step reversal observed in imaging remains invisible in the spatially integrated hysteresis loops. When complemented by precise tuning of the external magnetic fiel…
In this paper we propose a dark-state-based trapping strategy to break the optical diffraction limit for microscopy. We utilize a spatially dependent coupling field and a probe laser field with temporal and spatial modulation to interact with three-level atoms. The temporal modulation allows us to reduce the full width at half maximum (FWHM) of point spread function, and the spatial modulation help us obtain better spatial resolution than Gaussian beam. In addition, we also propose a proof-of-principle experiment protocol and discuss its feasibility.
In this paper, we review spatial light interference microscopy (SLIM), a common-path, phase-shifting interferometer, built onto a phase-contrast microscope, with white-light illumination. As one of the most sensitive quantitative phase imaging (QPI) methods, SLIM allows for speckle-free phase reconstruction with sub-nanometer path-length stability. We first review image formation in QPI, scattering, holography, and microcopy. Then, we outline SLIM imaging from theory to instrumentation. Zernike phase-contrast microscopy, phase retrieval in SLIM, and halo removal algorithms are discussed. Next,…
We report a method to measure the size of single dielectric nanoparticles with high accuracy and precision using quantitative differential interference contrast (DIC) microscopy. Dielectric nanoparticles are detected optically by the conversion of the optical phase change into an intensity change using DIC. Phase images of individual nanoparticles were retrieved from DIC by Wiener filtering, and a quantitative methodology to extract nanoparticle sizes was developed. Using polystyrene beads of 100 nm radius as size standard, we show that the method determines this radius within a few nm accurac…
