Real-time Quantitative Phase Imaging Research Articles

Deep-learning based flat-fielding quantitative phase contrast microscopy.

Quantitative phase contrast microscopy (QPCM) can realize high-quality imaging of sub-organelles inside live cells without fluorescence labeling, yet it requires at least three phase-shifted intensity images. Herein, we combine a novel convolutional neural network with QPCM to quantitatively obtain the phase distribution of a sample by only using two phase-shifted intensity images. Furthermore, we upgraded the QPCM setup by using a phase-type spatial light modulator (SLM) to record two phase-shifted intensity images in one shot, allowing for real-time quantitative phase imaging of moving samples or dynamic processes. The proposed technique was demonstrated by imaging the fine structures and fast dynamic behaviors of sub-organelles inside live COS7 cells and 3T3 cells, including mitochondria and lipid droplets, with a lateral spatial resolution of 245 nm and an imaging speed of 250 frames per second (FPS). We imagine that the proposed technique can provide an effective way for the high spatiotemporal resolution, high contrast, and label-free dynamic imaging of living cells.

Development of Robust On-Demand Droplet Generation System Using 3-D Image Reconstruction as Feedback

Droplet-based microfluidics is an emerging interdisciplinary field with broad applications. On-demand droplet generation with precise size allows performing sensitive detection and quantitative analysis with high throughput and separate units, while the essential precondition is characterizing the volume of droplet accurately. Generally, fluorescence or bright-field imaging can be adopted to provide the length or width of the droplet from 2-D images, and then derive an idealized model to approximately estimate the droplet volume, which would always lead to steady-state errors with ignoring the irregular curvature of droplet surface in the depth direction. In this work, we propose the usage of real-time quantitative phase imaging (QPI) technique for the 3-D detection of flowing droplets to provide a precise measurement of droplet volume which can serve as feedback signal. Comparative experiments with a passive droplet generation system confirm that our proposed method can offer higher accuracy and monodispersity of on-demand droplet generation, together with enhanced stability and anti-interference to resist internal or external perturbations, which could promise a predictable outcome in many microfluidics-based systems.

Single-shot color-coded LED microscopy for quantitative differential phase contrast imaging

Common quantitative differential phase contrast microscopy imaging usually uses multistep circular or annular illuminations and color multiplexed illuminations, bringing about low capture efficiency and poor phase reconstruction, respectively. We present a new single-shot quantitative differential phase contrast microscopy imaging method, which uses R/B-G annular LED multiplexed illumination to achieve better phase reconstruction by capturing 1 colored image. By deriving the phase transfer function of the illumination and simulation verification, the superiority of it is confirmed. Through phase reconstruction simulations for circular step and gradual samples, we verify that the R/B-G annular multiplexed illumination has better axial phase reconstruction accuracy and weaker lateral anisotropy than other color multiplexed illuminations. As a demonstration, quantitative phase imaging is performed on Quantitative Phase Target, validating the axial accuracy, lateral resolution, and lateral anisotropy of our method. Imaging and phase reconstruction were also performed on mouse kidney tissue slice and motional silica microspheres to visually express the utility and practicability of this method. Our research provides a better color multiplexed avenue for the real-time quantitative phase imaging in dispersion-free, weak-phase samples, especially living biological tissues.

Model-based feedback control for on-demand droplet dispensing system with precise real-time phase imaging

Recent years have witnessed the advances of droplet-based microfluidics as a powerful tool for high-throughput assays in biology, chemistry, material and environmental sciences. Precise and effective droplet generation is critical for the practical applications of droplet-based microfluidics. Although many passive and active droplet generation methods have been proposed and demonstrated, there are still challenges towards the implementation of droplet-on-demand in terms of system complexity, precision and robustness. In this work, we propose and demonstrate a dual-pressure-pulse (DPP) driven droplet generation method incorporated with model-based feedback control to achieve on-demand droplet generation. Based on the study of droplet formation dynamics in a flow-focusing configuration using real-time quantitative phase imaging (QPI) technique, we validate the proposed dual-pressure-pulse (DPP) driven droplet generation concept, for which the physical model of droplet formation can be greatly simplified. With this approach, model-based feedback control can be implemented for DPP actuation to achieve on-demand droplet generation with relatively high system response and low steady-state error.

Single-shot x-ray phase-contrast and dark-field imaging based on coded binary phase mask

We introduce a coded-mask-based multi-contrast imaging method for high-resolution phase-contrast and dark-field imaging. The method uses a binary phase mask designed to provide an ultra-high-contrast pattern and reference-free single-shot measurement and an algorithm based on maximum-likelihood optimization and automatic differentiation to perform simultaneous reconstruction of absorption, phase, and dark-field object images. Further, we demonstrate that the method has great potential for real-time quantitative phase imaging and wavefront sensing when combined with deep learning.

Real-time quantitative phase imaging by single-shot dual-wavelength off-axis digital holographic microscopy.

A single-shot dual-wavelength digital holographic microscopy with an adjustable off-axis configuration is presented, which helps realize real-time quantitative phase imaging for living cells. With this configuration, two sets of interference fringes corresponding to their wavelengths can be flexibly recorded onto one hologram in one shot. The universal expression on the dual-wavelength hologram recorded under any wave vector orientation angles of reference beams is given. To avoid as much as possible the effect of zero-order spectrum, we can flexibly select their carry frequencies for the two wavelengths using this adjustable off-axis configuration, according to the distribution feature of object's spatial-frequency spectrum. This merit is verified by a quantitative phase imaging experiment for the microchannel of a microfluidic chip. The reconstructed phase maps of living onion epidermal cells exhibit cellular internal life activities, for the first time to the best of our knowledge, vividly displaying the progress of the nucleus, cell wall, cytoskeleton, and the substance transport in microtubules inside living cells. These imaging results demonstrate the availability and reliability of the presented method for real-time quantitative phase imaging.

Off-axis tilt compensation in common-path digital holographic microscopy based on hologram rotation.

We present a simple and effective compensation method for the off-axis tilt in common-path digital holographic microscopy (CPDHM) by introducing a rotating operation on the hologram. The proposed method mainly requires a digital reference hologram (DRH), which is a rotated version of the original one; it is assumed to be easy to obtain by rotating the specimen's hologram 180°. In this way, the off-axis tilt could be removed by subtracting the retrieved phase of DRH from the retrieved phase of the original hologram, but without any complex spectrum centering judgment, fitting procedures, or prior knowledge of the system. This highly automatic and efficient performance makes our approach available for real-time quantitative phase imaging (QPI), although it limits the field of view (FOV) of the specimen. Some experimental results of microlens array and phase plate demonstrate the feasibility and effectiveness of the proposed method.

Quadriwave lateral shearing interferometric microscopy with wideband sensitivity enhancement for quantitative phase imaging in real time

Real-time quantitative phase imaging has tremendous potential in investigating live biological specimens in vitro. Here we report on a wideband sensitivity-enhanced interferometric microscopy for quantitative phase imaging in real time by employing two quadriwave lateral shearing interferometers based on randomly encoded hybrid gratings with different lateral shears. Theoretical framework to analyze the measurement sensitivity is firstly proposed, from which the optimal lateral shear pair for sensitivity enhancement is also derived. To accelerate the phase retrieval algorithm for real-time visualization, we develop a fully vectorized path-independent differential leveling phase unwrapping algorithm ready for parallel computing, and the framerate for retrieving the phase from each pair of two 4 mega pixel interferograms is able to reach 47.85 frames per second. Experiment results demonstrate that the wideband sensitivity-enhanced interferometric microscopy is capable of eliminating all the periodical error caused by spectral leaking problem and reducing the temporal standard deviation to the half level compared with phase directly retrieved by the interferogram. Due to its high adaptability, the wideband sensitivity-enhanced interferometric microscopy is promising in retrofitting existing microscopes to quantitative phase microscopes with high measurement precision and real-time visualization.

Real-time quantitative phase imaging based on transport of intensity equation with dual simultaneously recorded field of view.

Since quantitative phase distribution reflects both cellular shapes and conditions from another view, compared to traditional intensity observation, different quantitative phase microscopic methods are proposed for cellular detections. However, the transport of intensity equation-based approach not only presents phase, but also intensity, which attracts much attention. While classical transport of intensity equation needs multi-focal images which often cannot realize simultaneous phase measurement, in this Letter, to break through the limitation, a real-time quantitative phase imaging method using transport of intensity equation is proposed. Two identical CCD cameras are set at the binocular tubes to capture the same field of view but at different focal planes. With a double-frame algorithm assuming that the on-focal image is the average of over- and under-focal information, the proposed method is capable of calculating quantitative phase distributions of samples accurately and simultaneously indicating its potentialities in cellular real-time monitoring.

Measuring the Nonuniform Evaporation Dynamics of Sprayed Sessile Microdroplets with Quantitative Phase Imaging.

We demonstrate real-time quantitative phase imaging as a new optical approach for measuring the evaporation dynamics of sessile microdroplets. Quantitative phase images of various droplets were captured during evaporation. The images enabled us to generate time-resolved three-dimensional topographic profiles of droplet shape with nanometer accuracy and, without any assumptions about droplet geometry, to directly measure important physical parameters that characterize surface wetting processes. Specifically, the time-dependent variation of the droplet height, volume, contact radius, contact angle distribution along the droplet's perimeter, and mass flux density for two different surface preparations are reported. The studies clearly demonstrate three phases of evaporation reported previously: pinned, depinned, and drying modes; the studies also reveal instances of partial pinning. Finally, the apparatus is employed to investigate the cooperative evaporation of the sprayed droplets. We observe and explain the neighbor-induced reduction in evaporation rate, that is, as compared to predictions for isolated droplets. In the future, the new experimental methods should stimulate the exploration of colloidal particle dynamics on the gas-liquid-solid interface.

Optical Volume Measurement of Beating Cardiac Myocytes using Quantitative Phase Imaging

This paper describes recent research in developing a high-resolution optical microscope capable of real-time quantitative phase imaging to study morphological changes such as relative optical volume and optical thickness in cellular structures. A pixelated wire grid polarizer mask bonded to a CCD camera sensor enables simultaneous measurement of multiple phase contrast interference patterns. These data are processed in parallel to obtain phase, optical path difference (OPD), optical thickness (OT), optical volume (OV), simulated DIC (gradient), simulated dark field (gradient magnitude), and 3D typographic movies.Results will be presented of quantitative measurements of optical volume changes in beating rat cardiac myocytes before and after treatment with IPHC (isoproterenol hydrochloride). Full phase and topographic data were obtained at 15 frames/sec. Live cells were prepared and grown on either #1 coverslips or highly reflective slides. Cells cultures were placed into a Bioptechs FCS3 perfusion chamber and kept at 37°C during measurement. Videos of cell optical thickness topography were generated and processed to obtain relative optical volume. These results show dramatically increased rate and strength of contractions with application of IPHC.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Real-time quantitative phase imaging in biomedicine

Real-time quantitative phase imaging in biomedicine

Real-time quantitative phase imaging with a spatial phase-shifting algorithm

This Letter reports on the use of a spatial phase-shifting algorithm in a fast, straightforward method of real-time quantitative phase imaging. The computation time for phase extraction is five times faster than a Fourier transform and twice as fast as a Hilbert transform. The fact that the phase extraction from an interferogram of 512 × 512 pixels takes less than 8.93 ms with a typical desktop computer suggests the proposed method can be readily applied to high-speed dynamic quantitative phase imaging. The proposed method of quantitative phase imaging is effective and sufficiently general for application to the dynamic phenomena of biological samples.