Quantitative phase microscopy (QPM), a technique combining phase imaging and microscopy, enables visualization of the 3D topography in reflective samples, as well as the inner structure or refractive index distribution of transparent and translucent samples. Similar to other imaging modalities, QPM is constrained by the conflict between numerical aperture (NA) and field of view (FOV): an imaging system with a low NA has to be employed to maintain a large FOV. This fact severely limits the resolution in QPM up to being the illumination wavelength. Consequently, finer structures of samples cannot be resolved by using modest NA objectives in QPM. Aimed to that, many approaches, such as oblique illumination, structured illumination, and speckle illumination (just to cite a few), have been proposed to improve the spatial resolution (or the space bandwidth product) in phase microscopy by restricting other degrees of freedom (mostly time). This paper aims to provide an up to date review on the resolution enhancement approaches in QPM, discussing the pros and cons of each technique as well as the confusion on resolution definition claims on QPM and other coherent microscopy methods. Through this survey, we will review the most appealing and useful techniques for superresolution in coherent microscopy, working with and without lenses and with special attention to QPM.
High-Fidelity Single-Shot Quantitative Differential Phase Microscopy Using Pseudothermal Sagnac Interferometer
UiT The Arctic University of Norway（挪威阿尔塔大学）；Warsaw University of Technology, Institute of Micromechanics and Photonics（华沙理工大学微机械与光子学研究所）；Karolinska Institute（卡罗林斯卡学院）；The Faculty of Mathematics and Natural Sciences, Department of Physics, University of Oslo（奥斯陆大学数学与自然科学学院物理系）
In this paper we present a method to robustly evaluate the quantitative accuracy of various tomographic phase microscopy (TPM) methods with a multiple scattering 3D-printed microphantom with known geometry and refractive index distribution. We demonstrate this method by fabricating a multiple-scattering phantom and comparing 3D refractive index results that are output from three TPM reconstruction methods operating with visible and near-infrared wavelengths. One of these methods assumes the sample to be weak-scattering, while the other two take multiple scattering into account. This study can …
We suggest a new multimodal imaging technique for quantitatively measuring the integral (thickness-average) refractive index of the nuclei of live biological cells in suspension. For this aim, we combined quantitative phase microscopy with simultaneous 2-D fluorescence microscopy. We used 2-D fluorescence microscopy to localize the nucleus inside the quantitative phase map of the cell, as well as for measuring the nucleus radii. As verified offline by both 3-D confocal fluorescence microscopy and by 2-D fluorescence microscopy while rotating the cells during flow, the nucleus of cells in suspe…
We present a new technique for obtaining simultaneous multimodal quantitative phase and fluorescence microscopy of biological cells, providing both quantitative phase imaging and molecular specificity using a single camera. Our system is based on an interferometric multiplexing module, externally positioned at the exit of an optical microscope. In contrast to previous approaches, the presented technique allows conventional fluorescence imaging, rather than interferometric off-axis fluorescence imaging. We demonstrate the presented technique for imaging fluorescent beads and live biological cel…
Hyperspectral stimulated Raman scattering (SRS) microscopy is a label-free technique for biomedical and mineralogical imaging which can suffer from low signal to noise ratios. Here we demonstrate the use of an unsupervised deep learning neural network for rapid and automatic denoising of SRS images: UHRED (Unsupervised Hyperspectral Resolution Enhancement and Denoising). UHRED is capable of one-shot learning; only one hyperspectral image is needed, with no requirements for training on previously labelled datasets or images. Furthermore, by applying a k-means clustering algorithm to the process…
In this work, we present a highly efficient quantitative phase imaging (QPI) approach using programmable annular LED illumination based on traditional bright-field microscope. As a new type of coded illumination, the LED array provides a flexible and compatible way to realize QPI. The proposed method modulates the transfer function of system by changing the LED illumination pattern, which achieves twice resolution of objective NA and gives noise-robust response of transfer function. The phase of a sample could be recovered from the intensity images with the inversion of transfer function. More…
We present geometric-phase microscopy allowing a multipurpose quantitative phase imaging in which the ground-truth phase is restored by quantifying the phase retardance. The method uses broadband spatially incoherent light that is polarization sensitively controlled through the geometric (Pancharatnam-Berry) phase. The assessed retardance possibly originates either in dynamic or geometric phase and measurements are customized for quantitative mapping of isotropic and birefringent samples or multi-functional geometric-phase elements. The phase restoration is based on the self-interference of po…
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…
The principles of quantum optics have yielded a plethora of ideas to surpass the classical limitations of sensitivity and resolution in optical microscopy. While some ideas have been applied in proof-of-principle experiments, imaging a biological sample has remained challenging mainly due to the inherently weak signal measured and the fragility of quantum states of light. In principle, however, these quantum protocols can add new information without sacrificing the classical information and can therefore enhance the capabilities of existing super-resolution techniques. Image scanning microscop…
Differential phase contrast microscopy (DPC) provides high-resolution quantitative phase distribution of thin transparent samples under multi-axis asymmetric illuminations. Typically, illumination in DPC microscopic systems is designed with 2-axis half-circle amplitude patterns, which, however, result in a non-isotropic phase contrast transfer function (PTF). Efforts have been made to achieve isotropic DPC by replacing the conventional half-circle illumination aperture with radially asymmetric patterns with 3-axis illumination or gradient amplitude patterns with 2-axis illumination. Neverthele…
Impact of refractive index heterogeneity on stimulated Brillouin scattering microscopy: a quantitative analysis
Shanghai Institute of Optics and Fine Mechanics, Chinese Academy of Sciences（中国科学院上海光学精密机械研究所）；University of Chinese Academy of Sciences（中国科学院大学）；European Molecular Biology Laboratory（欧洲分子生物学实验室）
We present a technically simple implementation of quantitative phase imaging in confocal microscopy based on synthetic optical holography with sinusoidal-phase reference waves. Using a Mirau interference objective and low-amplitude vertical sample vibration with a piezo-controlled stage, we record synthetic holograms on commercial confocal microscopes (Nikon, model: A1R; Zeiss: model: LSM-880), from which quantitative phase images are reconstructed. We demonstrate our technique by stain-free imaging of cervical (HeLa) and ovarian (ES-2) cancer cells and stem cell (mHAT9a) samples. Our techniqu…
White light phase-shifting interference microscopy (WL-PSIM) is a prominent technique for high-resolution quantitative phase imaging (QPI) of industrial and biological specimens. However, multiple interferograms with accurate phase-shifts are essentially required in WL-PSIM for measuring the accurate phase of the object. Here, we present single-shot phase-shifting interferometric techniques for accurate phase measurement using filtered white light phase-shifting interference microscopy (F-WL-PSIM) and deep neural network (DNN). The methods are incorporated by training the DNN to generate 1) fo…
Wavelength and Polarization Multiplexed Nonlocal Metasurface for Quantitative Phase Microscopy
ARC Centre of Excellence for Transformative Meta-Optical Systems, School of Physics, The University of Melbourne（墨尔本大学变革性超光学系统卓越中心物理学院）；School of Physics, The University of Melbourne（墨尔本大学物理学院）；Department of Biochemistry and Pharmacology, The University of Melbourne（墨尔本大学生化与药理学系）；Functional Materials and Microsystems Research Group and the Mico Nano Research Facility, RMIT University（皇家墨尔本理工大学功能材料与微系统研究组及微电子纳米研究设施）；ARC Centre of Excellence for Transformative Meta-Optical Systems, RMIT University（皇家墨尔本理工大学变革性超光学系统卓越中心）
Quantitative phase microscopy (QPM) has found significant applications in the field of biomedical imaging which works on the principle of interferometry. The theory behind achieving interference in QPM with conventional light sources such as white light and lasers is very well developed. Recently, the use of dynamic speckle illumination (DSI) in QPM has attracted attention due to its advantages over conventional light sources such as high spatial phase sensitivity, single shot, scalable field of view (FOV) and resolution. However, the understanding behind obtaining interference fringes in QPM …
Optical metasurfaces have emerged as a new generation of building blocks for multi-functional optics. Design and realization of metasurface elements place ever-increasing demands on accurate assessment of phase alterations introduced by complex nanoantenna arrays, a process referred to as quantitative phase imaging. Despite considerable effort, the widefield (non-scanning) phase imaging that would approach resolution limits of optical microscopy and indicate the response of a single nanoantenna still remains a challenge. Here, we report on a new strategy in incoherent holographic imaging of me…
In this paper, we describe the implementation of leakage radiation microscopy (LRM) to probe the chirality of plasmonic nanostructures. We demonstrate experimentally spin-driven directional coupling as well as vortex generation of surface plasmon polaritons (SPPs) by nanostructures built with T-shaped and $Λ$- shaped apertures. Using this far-field method, quantitative inspections, including directivity and extinction ratio measurements, are achieved via polarization analysis in both image and Fourier planes. To support our experimental findings, we develop an analytical model based on a multi…
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…
Lanthanide-based upconversion nanoparticles (UCNPs) generally require high power laser excitation. Here we report wide-field upconversion microscopy at single-nanoparticle sensitivity using incoherent excitation of a 970-nm light-emitting diode (LED). We show that due to its broad emission spectrum, LED excitation is about 3 times less effective for UCNPs and generates high background compared to laser illumination. To counter this, we use time-gated luminescence detection to eliminate the residual background from the LED source, so that individual UCNPs with high sensitizer (Yb3+) doping and …
Quantum enhanced microscopy allows for measurements at high sensitivities and low damage. Recently, multi-pass microscopy was introduced as such a scheme, exploiting the sensitivity enhancement offered by multiple photon-sample interactions. Here we theoretically and numerically compare three different contrast enhancing techniques that are all based on self-imaging cavities: CW cavity enhanced microscopy, cavity ring-down microscopy and multi-pass microscopy. We show that all three schemes can lead to sensitivities beyond the standard quantum limit.
We report thorough measurements of surface plasmon polaritons (SPPs) running along nearly perfect air-gold interfaces formed by atomically flat surfaces of chemically synthesized gold monocrystals. By means of amplitude- and phase-resolved near-field microscopy, we obtain their propagation length and effective mode index at visible wavelengths (532, 594, 632.8, 729, and 800 nm). The measured values are compared with the values obtained from the dielectric functions of gold that are reported in literature. Importantly, a reported dielectric function of monocrystalline gold implies $\sim 1.5$ ti…
Quadriwave lateral shearing interferometry (QLSI) is a quantitative phase imaging technique based on the use of a diffraction grating placed in front of a camera. This grating creates a wire-mesh-like image, called an interferogram, that is postprocessed to retrieve both the intensity and phase profiles of an incoming light beam. Invented in the 90s, QLSI has been used in numerous applications, e.g., laser beam characterization, lens metrology, topography measurements, adaptive optics, or gas jet metrology. More recently, the technique has been implemented on optical microscopes to characteriz…
For quantitative phase imaging (QPI) based on transport-of-intensity equation (TIE), partially coherent illumination provides speckle-free imaging, compatibility with brightfield microscopy, and transverse resolution beyond coherent diffraction limit. Unfortunately, in a conventional microscope with circular illumination aperture, partial coherence tends to diminish the phase contrast, exacerbating the inherent noise-to-resolution tradeoff in TIE imaging, resulting in strong low-frequency artifacts and compromised imaging resolution. Here, we demonstrate how these issues can be effectively add…
Quantitative phase gradient microscopy with spatially entangled photons
University of Ottawa（渥太华大学）；National Research Council of Canada（加拿大国家研究委员会）；Joint Centre for Extreme Photonics（极端光子学联合中心）；National Cheng Kung University（国立成功大学）；Center for Quantum Frontiers of Research and Technology, NCKU（国立成功大学量子前沿研究与技术中心）；Institute for Quantum Studies, Chapman University（查普曼大学量子研究所）
Changes in morphological characteristics of cells from two cultured cancer cell lines, HeLa and A549, induced by photodynamic treatment with Radachlorin photosensitizer have been monitored using digital holographic microscopy. The observed dose-dependent post-treatment dynamics of phase shift variations demonstrated several scenarios of cell death. In particular the phase shift increase at low doses can be associated with apoptosis while its decrease at high doses can be associated with necrosis. Two cell types were shown to be differently responsive to treatment at the same doses. Although th…
Raman spectroscopy reveals chemically specific information and provides label-free insight into the molecular world. However, the signals are intrinsically weak and call for enhancement techniques. Here, we demonstrate Purcell enhancement of Raman scattering in a tunable high-finesse microcavity, and utilize it for molecular diagnostics by combined Raman and absorption imaging. Studying individual single-wall carbon nanotubes, we identify crucial structural parameters such as nanotube radius, electronic structure and extinction cross-section. We observe a 320-times enhanced Raman scattering sp…
Ultrafast Electron Microscopy (UEM) has been demonstrated to be an effective table-top technique for imaging the temporally-evolving dynamics of matter with subparticle spatial resolution on the time scale of atomic motion. However, imaging the faster motion of electron dynamics in real time has remained beyond reach. Here, we demonstrate more than an order of magnitude (16 times) enhancement in the typical temporal resolution of UEM by generating isolated 30 fs electron pulses, accelerated at 200 keV, via the optical-gating approach, with sufficient intensity for efficiently probing the elect…
Tissue biopsy evaluation in the clinic is in need of quantitative disease markers for diagnosis and, most importantly, prognosis. Among the new technologies, quantitative phase imaging (QPI) has demonstrated promise for histopathology because it reveals intrinsic tissue nanoarchitecture through the refractive index. However, a vast majority of past QPI investigations have relied on imaging unstained tissues, which disrupts the established specimen processing. Here we present color spatial light interference microscopy (cSLIM) as a new whole slide imaging modality that performs interferometric …
Plasmonic nanoparticles influence the absorption and emission processes of nearby emitters due to local enhancements of the illuminating radiation and the photonic density of states. Here, we use the plasmon resonance of metal nanoparticles in order to enhance the stimulated depletion of excited molecules for super-resolved microscopy. We demonstrate stimulated emission depletion (STED) microscopy with gold nanorods with a long axis of only 26 nm and a width of 8 nm that provide an enhancement of the resolution compared to fluorescent-only probes without plasmonic components irradiated with th…
Duke University（杜克大学）；University of California San Diego（加州大学圣地亚哥分校）；Duke University Medical Center（杜克大学医学中心）
We report Tensorial Tomographic Differential Phase-Contrast microscopy (T2DPC), a quantitative label-free tomographic imaging method for simultaneous measurement of phase and anisotropy. T2DPC extends differential phase-contrast microscopy, a quantitative phase imaging technique, to highlight the vectorial nature of light. The method solves for permittivity tensor of anisotropic samples from intensity measurements acquired with a standard microscope equipped with an LED matrix, a circular polarizer, and a polarization-sensitive camera. We demonstrate accurate volumetric reconstructions of refr…
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…
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 sens…
Deep neural networks have emerged as effective tools for computational imaging including quantitative phase microscopy of transparent samples. To reconstruct phase from intensity, current approaches rely on supervised learning with training examples; consequently, their performance is sensitive to a match of training and imaging settings. Here we propose a new approach to phase microscopy by using an untrained deep neural network for measurement formation, encapsulating the image prior and imaging physics. Our approach does not require any training data and simultaneously reconstructs the soug…
