Partial coherent imaging, which provides high robustness and twice the imaging resolution of the coherent diffraction limit, has become a hot research method in quantitative phase imaging. Asymmetric illumination is one of the most common methods to generate phase contrast for weakly absorbed samples. By establishing a strict intensity-phase model, the quantitative phase distribution of the sample is then obtained by inverse algorithm. In order to linearize the imaging process, weak phase approximation, which imposes restrictions of small value phase on sample, is introduced into the partially coherent imaging model to separate the sample absorption and phase. However, the weak phase approximation introduces an uncertain phase loss in quantitative phase imaging, especially for samples with a large phase. In this paper, we investigate the quantitative definition weak phase approximation for partial coherent quantitative phase imaging under asymmetric illumination by simulations. According to the simulation results, we find that the reconstruction accuracy of the weak phase approximation is not only determined by the absolute phase value of the sample, but also a
effected by the illumination aperture. Furthermore, a quantitative definition of the weak phase approximation is given to provide a basis for the phase reconstruction accuracy for quantitative phase imaging based on asymmetric illumination.
According to the phase gradient transfer function (PGTF) derived from the phase space theory, the phase recovery algorithm based on the transport of intensity equation (TIE) has the problem that the high-frequency phase is underestimated due to the coherence effect of the limited aperture system under partially coherent illumination. Therefore, based on the theory of PGTF and phase transfer function (PTF), a phase reconstruction algorithm named high-resolution synthetic spectrum (HSS) method combining the TIE and the PTF-based deconvolution is proposed. This technique broadens the application range and provides high contrast, high accuracy, and highresolution quantitative phase results with high robustness. The performances of this technology are demonstrated by simulation and experiments, showing efficient for phase retrieval in the near-Fresnel region. Such a highresolution method can offer a flexible and cost-effective alternative for biomedical research and cell analysis, providing new avenues to design powerful computational imaging systems
Differential phase contrast (DPC) imaging is a popular spatially partially coherent imaging method, which pro- vides high-quality, speckle-free 3D reconstructions with lateral resolution up to twice the coherent diffraction limit, under the precondition that the pixel size of the imaging sensor is small enough to prevent spatial alias- ing/undersampling. However, cameras are in general designed to have a large pixel size so that the intensity information transmitted by the optical system cannot be adequately sampled or digitized. On the other hand, using an image sensor with smaller pixel size or adding a magnification camera adapter to the camera can re- solve the undersampling at the expense of a reduced field of view (FOV). To solve this tradeoff, we introduce a new variation of quantitative DPC approach, termed anti-aliased DPC (AADPC), which uses several aliased intensity images under asymmetric illuminations to recover wide-field aliasing-free phase images. AADPC starts from an initial phase estimate obtained by a DPC-like deconvolution based on the systems weak phase transfer function. Then the obtained initial phase map is further refined by the iterative de-multiplexing algorithm to overcome pixel-aliasing and improve the imaging resolution. The data redundancy requirement as well as the optimal illumination scheme of AADPC are analyzed and discussed, suggesting the spatial undersampling can be mitigated through the iterative algorithm that uses only 4 images, yielding a nearly 4-fold increase in the space-bandwidth product (SBP) compared to conventional DPC approach.
We demonstrate a method for increasing the effective resolution of phase retrieval based on the transport of intensity equation (TIE) named speckle high-resolution synthetic spectrum (speckle-HSS), as the upgraded version of the speckle-TIE approach we proposed before based on the quantitative phase imaging camera with a weak diffuser (QPICWD). Benefit from the phase gradient transfer function (PGTF) and phase transfer function (PTF), the phase blurring caused by the underestimation of phase gradient can be compensated correctly via combining TIE and PTF-based deconvolution. This method broadens the application range, alleviating the artifacts and enhancing the contrast and resolution in more accurate value. The experimental results of live HeLa cells have been presented, showing the effectiveness of the proposed method.
A commercialized digital holographic microscope with complete software supporting is composed of software and hardware, and also has quantitative phase recovery and phase compensation algorithm support. The calculation speed can reach thirty frames per second, and the resolution can reach 0.775 μm under a 20 times 0.4 NA objective lens, which can realize subcellular three-dimensional reconstruction and thickness distribution measurement of transparent samples in real-time and high resolution. This paper describes the optical path geometry, algorithm flow and image calibration of the system, including the phase aberration compensation algorithm based on principal component analysis (PCA), unwrapping algorithm based on reliability analysis and subpixel displacement technology. These algorithms are processed by software in real time and can be adjusted flexibly. In addition, the whole system is tested by using phase resolution board. Quantitative phase recovery, three-dimensional structure display and differential interference contrast (DIC) display were performed for human cervical cancer cells and human oral epithelial cells, and quantitative profile measurement was conducted for pollen.
We present a miniaturized microscopic imaging system to achieve multi-contrast label-free imaging. In our imaging system, a highly integrated optical system using a miniaturization lens with fixed focal length replaces the complex optical path of traditional microscope systems, significantly reducing the size of the microscope to 14*16.5*20 cm3 . A programmable full-color light emitting diode (LED), which is controlled by independently designed operating software, is used to illuminate the sample in different illumination patterns. In addition, we developed a QT-based operating software to implement the synchronous control of the hardware system to achieve various label-free imaging approaches, including bright field, dark field, rainbow dark field, Rheinberg, differential phase contrast, and quantitative phase imaging. All the microscopic imaging approaches and system parameters can be controlled by the software without any hardware modification. In addition, some functions of the cell analysis are also added to our system, which can realize cell counter, 3D information measurement. We demonstrated our miniaturized system and its imaging results through experiments, which showed that our system is simple and fast to operate, providing more visual imaging results and high-resolution 3D information for samples. The experiment on living cell demonstrated that our miniaturized system can be built into an incubator for imaging and analysis of living cells in an environment suitable for cell growth.
In this paper, a holographic lensless quantitative phase imaging (QPI) microscope is presented, which is composed of a CMOS detector image sensor with a programmable color LED matrix, without any lens and mechanical displacement device. Such a miniaturized system can provide a field-portable cost-effective platform for highthroughput quantification of multiple samples. Coordinating the self-developed software operating system, the bright-field imaging, the quantitative phase imaging as well as cell counting, profile analysis, three-dimensional (3D) imaging and differential interference contrast (DIC) imaging can be realized. With its high-resolution based computational microscopy interface, this system can be also adaptively used for telemedicine applications and point-of-care testing (POCT) in resource-limited environments.
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, reduce the temporal resolution of DPC, precluding observation of high-speed phenomenon. Efforts have been made to achieve video-rate DPC by using tri-mode illumination or adding multi-colored filter. However, the frequency responses of the PTFs has not been improved, leading to poor phase contrast and signal-to-noise ratio (SNR) for phase reconstruction. We present a video-rate isotropic quantitative phase imaging (QPI) method based on color-multiplexed differential phase contrast (DPC). In our method, the illumination source is modulated by an LCD into an annular color-multiplexed pattern matching the numerical aperture of the objective precisely to maximize the frequency response for both low and high frequencies (from 0 to 2NAobj). In addition, we propose an alternating illumination scheme to provide a perfectly circularly symmetrical phase transfer function (PTF), achieving isotropic imaging resolution and signal-to-noise ratio (SNR). A color camera records the light transmitted through the specimen, and three monochromatic intensity images at each color channel are then separated and utilized to recover the phase of the specimen. We present the derivation, implementation, simulation and experimental results demonstrating that our method accomplishes high resolution, noise-robustness and reconstruction accuracy at camera-limited frame rates.
We present a novel approach to compensate coherence effect via combining the transport of intensity equation (TIE) with look-up table phase compensation (LUT-PC) method. It is the better version of the Speckle-TIE method we demonstrated before on the basis of the quantitative phase imaging camera with a weak diffuser (QPICWD). With the phase gradient ratio theory and the look-up table method, the phase blurring caused by underestimation of phase gradient will be compensated correctly by reasonable rescaling. The LUT-PC SpeckleTIE method has the evident predominance of speediness since it only needs one slightly defocused speckle image in one time owing to that the reference speckle image can be captured beforehand. The deblurring achieved by this method improves the imaging resolution to the theoretical partial coherence limit with good robustness, reducing artifacts and improving the accuracy and contrast. The experimental results show the effectiveness of the technique.
Quantitative phase imaging (QPI), which provides unique imaging capabilities for optical thickness variation of living cells and tissues without the need for specific staining or exogenous contrast agents (e.g., dyes or fluorophores), has emerged as an invaluable optical tool for biomedical research. Differential phase contrast (DPC) is the most promising QPI approach to high resolution label-free cellular dynamic imaging because of its advantages of higher imaging efficiency, higher accuracy, and higher stability. Typically, illuminations in DPC systems are designed with 2-axis half-circle amplitude patterns, which however results in a non-isotropic phase transfer function (PTF). Furthermore, the frequency responses of the PTFs have not been fully optimized, leading to suboptimal phase contrast and signal-to-noise ratio (SNR) for phase reconstruction. In this paper, we derive the optimal illumination scheme to maximize the PTF response for both low and high frequencies (from 0 to 2NAobj ), and meanwhile achieve perfectly isotropic PTF with only 2-axis intensity measurements. We present the theoretical analysis, simulations, and experimental results demonstrating that our optimal illumination scheme is a simple, efficient, and stable approach for label-free quantitative cell imaging with subcellular resolution.
We present a new quantitative phase imaging method on the basis of the novel camera named quantitative phase imaging camera with a weak diffuser (QPICWD). It measures object under low-coherence quasi-monochromatic illumination via examining the deformation of the speckle intensity pattern. The speckle deformation can be analyzed by means of ensemble average of geometric flow method, realizing high resolution distortion field by using the transport of intensity equation (TIE). There are some applications for the proposed new design including nondestructive optical testing of microlens array with nanometric thickness. Since the proposed QPICWD needs no modification of the common bright-field microscope, it may promote QPI as a useful tool for subcellular level biological analysis.
We present an efficient quantitative phase imaging camera with a weak diffuser (QPICWD) based on the transport of intensity equation (TIE). The compact QPICWD measures object induced phase delay under low-coherence quasi-monochromatic illumination via examining the deformation of the speckle intensity pattern. Analysing the speckle deformation with an ensemble average of the geometric ow, we can achieve the high-resolution distortion field by the TIE. We present some applications for the proposed design involving nondestructive optical testing of microlens array with nanometric thickness and imaging of fixed and live unstained HeLa cells. Since the designed QPICWD needs no modification of the common bright-field microscope or additional accessories, it may advance QPI as a widely useful tool for biological analysis at subcellular levels.
Fourier ptychographic microscopy (FPM) is a wide-field and high-resolution (HR) imaging technique, reconstructing HR spectrum from a series of low-resolution (LR) images captured at different illumination angles. In FPM, the quality of captured images is a critical factor that affects the final reconstruction HR result, so an effective denoising method is an indispensable process step. Here we propose an adaptive denoising method for FPM, which takes advantage of the data redundancy of FPM to separate signal from noise without any pre-knowledge about the noise statistics. Different from the traditional denoising method by reducing a fixed threshold, the proposed adaptive denoising method can more effectively eliminate noise and preserve more effective signals. This paper explains adaptive denoising principle and process steps, and finally demonstrates that this method not only improve the accuracy and robustness of FPM, but also relax the imaging performance requirement for implementing high-quality FPM reconstruction.
Friendly interactive interface always significantly accelerate the progress of scientific research. However, most of the commercial softwares cannot meet the demand of a digital holographic microscope. Therefore, we designed a software in order to satisfy this requirement.
We use Visual Studio 2010 to build this software, which is based on MFC multi-documents and multi-threads. The main process of designing this software is as follow: 1) Firstly, build the main frame of the software. It is easy to realize the basic interface of Windows style by programming with MFC. The most important thing in this module is adding algorithms and the functions of tool buttons to the program. 2) Secondly, implement functionality of each sub module. In this software, sub modules mainly mean sub windows. In order to have the unity of style, all sub windows use the similar toolbar. Specially, if one sub window have its own functionality, we will add button alone. 3) Thirdly, pass messages among modules. Passing messages among modules is significant in this software. The news in main program must be transmitted to the relevant sub window. The operation information in any sub windows must be transmitted to the main program, or transmitted to other sub windows. In order to make the program more efficient, we utilize multi-thread programming. With a digital holographic microscope, our software has many useful features, such as capturing the hologram of a sample (Holo View), displaying its Fourier spectrum (Fourier View), unwrapping phase map (Phase View), digital refocusing intensity information (Intensity View), drawing 2D line across the sample (2D View) and displaying three-dimensional images (Plot View). The experimental results demonstrate that a digital holographic microscope could be used much easier with the help of our software.