High-resolution Quantitative Phase Imaging Research Articles

Resolution-enhanced quantitative phase imaging of blood platelets using a generative adversarial network

We developed a new method to enhance the resolution of blood platelet aggregates imaged via quantitative phase imaging (QPI) using a Pix2Pix generative adversarial network (GAN). First, 1 µm polystyrene beads were imaged with low- and high-resolution QPI, to train the GAN model and validate its applicability. Testing on the polystyrene beads demonstrated a mean error of 4.14% in the generated high-resolution optical-path-delay values compared to the optically acquired ones. Next, blood platelets were collected with low- and high-resolution QPI, and a deep neural network was trained to predict the high-resolution platelet optical-path-delay profiles using the low-resolution profiles, achieving a mean error of 7.01% in the generated high-resolution optical-path-delay values compared to the optically acquired ones. These results highlight the potential of the method in enhancing QPI resolution of cell aggregates without the need for sophisticated optical equipment and optical system modifications for high-resolution microscopy, allowing for better understanding of platelet-related disorders and conditions such as thrombocytopenia and thrombocytosis.

Fast Fourier ptychographic quantitative phase microscopy for in vitro label-free imaging.

Quantitative phase microscopy (QPM) is indispensable in biomedical research due to its advantages in unlabeled transparent sample thickness quantification and obtaining refractive index information. Fourier ptychographic microscopy (FPM) is among the most promising QPM methods, incorporating multi-angle illumination and iterative phase recovery for high-resolution quantitative phase imaging (QPI) of large cell populations over a wide field of-view (FOV) in a single pass. However, FPM is limited by data redundancy and sequential acquisition strategies, resulting in low imaging efficiency, which in turn limits its real-time application in in vitro label-free imaging. Here, we report a fast QPM based on Fourier ptychography (FQP-FPM), which uses an optimized annular downsampling and parallel acquisition strategy to minimize the amount of data required in the front end and reduce the iteration time of the back-end algorithm (3.3% and 4.4% of conventional FPM, respectively). Theoretical and data redundancy analyses show that FQP-FPM can realize high-throughput quantitative phase reconstruction at thrice the resolution of the coherent diffraction limit by acquiring only ten raw images, providing a precondition for in vitro label-free real-time imaging. The FQP-FPM application was validated for various in vitro label-free live-cell imaging. Cell morphology and subcellular phenomena in different periods were observed with a synthetic aperture of 0.75 NA at a 10× FOV, demonstrating its advantages and application potential for fast high-throughput QPI.

Structured illumination contrast transfer function for high resolution quantitative phase imaging.

We report a sub-diffraction resolution imaging of non-fluorescent samples through quantitative phase imaging. This is achieved through a novel application of structured illumination microscopy (SIM), a super-resolution imaging technique established primarily for fluorescence microscopy. Utilizing our contrast transfer function formalism with SIM, we extract the high spatial frequency components of the phase profile from the defocused intensity images, enabling the reconstruction of a quantitative phase image with a frequency spectrum that surpasses the diffraction limit imposed by the imaging system. Our approach offers several advantages including a deterministic, phase-unwrapping-free algorithm and an easily implementable, non-interferometric setup. We validate the proposed technique for high-resolution phase imaging through both simulation and experimental results, demonstrating a two-fold enhancement in resolution. A lateral resolution of 0.814 µm is achieved for the phase imaging of human cheek cells using a 0.42 NA objective lens and an illumination wavelength of 660 nm, highlighting the efficacy of our approach for high-resolution quantitative phase imaging.

Single-shot quantitative phase imaging with phase modulation of a liquid crystal spatial light modulator (LC-SLM) under white light illumination.

We propose a novel, to the best of our knowledge, single-shot quantitative phase imaging (QPI) technique with the phase modulation of a liquid crystal spatial light modulator (LC-SLM) under white light illumination. By studying the phase modulation characteristics of an LC-SLM under white light illumination, images captured at different wavelengths are equivalent to those captured at different defocus distances when loading a Fresnel lens pattern on the LC-SLM. Consequently, a color camera is able to simultaneously acquire multi-intensity images at different defocus distances. Finally, the phase is retrieved from a single-shot color image using the transport of intensity equation. To demonstrate the flexibility and accuracy of our method, a photoetched phase object and human red blood cells are quantitatively measured. An investigation of living yeast cells is conducted to verify the dynamic measurement capability. The proposed method provides a simple, efficient, and flexible means to accomplish real-time high-resolution quantitative phase imaging without sacrificing the field of view (FOV), which can be further integrated into a conventional microscope to achieve real-time microscopic QPI.

High-resolution single-shot phase-shifting interference microscopy using deep neural network for quantitative phase imaging of biological samples.

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 (520±36 nm) phase-shifting interference microscopy (F-WL-PSIM) and deep neural network (DNN). The methods are incorporated by training the DNN to generate (a) four phase-shifted frames and (b) direct phase from a single interferogram. The training of network is performed on two different samples i.e., optical waveguide and MG63 osteosarcoma cells. Further, performance of F-WL-PSIM+DNN framework is validated by comparing the phase map extracted from network generated and experimentally recorded interferograms. The current approach can further strengthen QPI techniques for high-resolution phase recovery using a single frame for different biomedical applications.

Accurate quantitative phase imaging by the transport of intensity equation: a mixed-transfer-function approach.

As a well-established deterministic phase retrieval approach, the transport of intensity equation (TIE) is able to recover the quantitative phase of a sample under coherent or partially coherent illumination with its through-focus intensity measurements. Nevertheless, the inherent paraxial approximation limits its validity to low-numerical-aperture imaging and slowly varying objects, precluding its application to high-resolution quantitative phase imaging (QPI). Alternatively, QPI can be achieved by phase deconvolution approaches based on the coherent contrast transfer function or partially coherent weak object transfer function (WOTF) without invoking paraxial approximation. But these methods are generally appropriate for "weakly scattering" samples in which the total phase delay induced by the object should be small. Consequently, high-resolution high-accuracy QPI of "nonweak" phase objects with fine details and large phase excursions remains a great challenge. In this Letter, we propose a mixed-transfer-function (MTF) approach to address the dilemma between measurement accuracy and imaging resolution. By effectively merging the phases reconstructed by TIE and WOTF in the frequency domain, the high-accuracy low-frequency phase "global" profile can be secured, and high-resolution high-frequency features can be well preserved simultaneously. Simulations and experimental results on a microlens array and unstained biological cells demonstrate the effectiveness of MTF.

High-resolution quantitative phase imaging based on a spatial light modulator and incremental binary random sampling.

We propose a single-beam high-resolution quantitative phase imaging method based on a spatial light modulator (SLM) and an incremental binary random sampling (IBRS) algorithm. In this method, the image of the test object presents on the image sensor through an optical microscopy system composed of an objective lens and a collimating lens. A transmittance SLM displaying a group of well-designed IBRS patterns is inserted in the optical microscopy system to modulate the object wavefront. The phase information of the object image can be quantitatively retrieved from the recorded intensities using the IBRS algorithm and the amplitude obtained directly from the diffraction intensity. The IBRS algorithm employed in our method has higher accuracy for phase retrieval compared with our previously proposed complementary random sampling algorithm, which is confirmed by simulations. Further, we demonstrate experimentally the feasibility of our method through several examples: phase imaging of immersion oil droplets with a diffraction-limited lateral resolution of 1.54 µm and a few microbiological specimens with 0.70 µm. Experimental results reveal that our proposed method provides a feasible single-beam technique for quantitative phase imaging with a high spatial resolution.

Unsupervised organization of cervical cells using bright-field and single-shot digital holographic microscopy.

We report results on unsupervised organization of cervical cells using microscopy of Pap-smear samples in brightfield (3-channel color) as well as high-resolution quantitative phase imaging modalities. A number of morphological parameters are measured for each of the 1450 cell nuclei (from 10 woman subjects) imaged in this study. The principal component analysis (PCA) methodology applied to this data shows that the cell image clustering performance improves significantly when brightfield as well as phase information is utilized for PCA as compared to when brightfield-only information is used. The results point to the feasibility of an image-based tool that will be able to mark suspicious cells for further examination by the pathologist. More importantly, our results suggest that the information in quantitative phase images of cells that is typically not used in clinical practice is valuable for automated cell classification applications in general.

High-Resolution Quantitative Phase Imaging of Plasmonic Metasurfaces with Sensitivity down to a Single Nanoantenna

High-Resolution Quantitative Phase Imaging of Plasmonic Metasurfaces with Sensitivity down to a Single Nanoantenna

High-Resolution Quantitative Phase Imaging of Plasmonic Metasurfaces with Sensitivity down to a Single Nanoantenna.

Optical metasurfaces have emerged as a new generation of building blocks for multifunctional 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 (nonscanning) 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 metasurfaces, in which unprecedented spatial resolution and light sensitivity are achieved by taking full advantage of the polarization selective control of light through the geometric (Pancharatnam-Berry) phase. The measurement is carried out in an inherently stable common-path setup composed of a standard optical microscope and an add-on imaging module. Phase information is acquired from the mutual coherence function attainable in records created in broadband spatially incoherent light by the self-interference of scattered and leakage light coming from the metasurface. In calibration measurements, the phase was mapped with the precision and spatial background noise better than 0.01 and 0.05 rad, respectively. The imaging excels at the high spatial resolution that was demonstrated experimentally by the precise amplitude and phase restoration of vortex metalenses and a metasurface grating with 833 lines/mm. Thanks to superior light sensitivity of the method, we demonstrated for the first time to our knowledge the widefield measurement of the phase altered by a single nanoantenna while maintaining the precision well below 0.15 rad.

Active intracellular transport in metastatic cells studied by spatial light interference microscopy.

Spatiotemporal patterns of intracellular transport are very difficult to quantify and, consequently, continue to be insufficiently understood. While it is well documented that mass trafficking inside living cells consists of both random and deterministic motions, quantitative data over broad spatiotemporal scales are lacking. We studied the intracellular transport in live cells using spatial light interference microscopy, a high spatiotemporal resolution quantitative phase imaging tool. The results indicate that in the cytoplasm, the intracellular transport is mainly active (directed, deterministic), while inside the nucleus it is both active and passive (diffusive, random). Furthermore, we studied the behavior of the two-dimensional mass density over 30 h in HeLa cells and focused on the active component. We determined the standard deviation of the velocity distribution at the point of cell division for each cell and compared the standard deviation velocity inside the cytoplasm and the nucleus. We found that the velocity distribution in the cytoplasm is consistently broader than in the nucleus, suggesting mechanisms for faster transport in the cytosol versus the nucleus. Future studies will focus on improving phase measurements by applying a fluorescent tag to understand how particular proteins are transported inside the cell.

Improved quantitative phase imaging in lensless microscopy by single-shot multi-wavelength illumination using a fast convergence algorithm.

We report on a novel algorithm for high-resolution quantitative phase imaging in a new concept of lensless holographic microscope based on single-shot multi-wavelength illumination. This new microscope layout, reported by Noom et al. along the past year and named by us as MISHELF (initials incoming from Multi-Illumination Single-Holographic-Exposure Lensless Fresnel) microscopy, rises from the simultaneous illumination and recording of multiple diffraction patterns in the Fresnel domain. In combination with a novel and fast iterative phase retrieval algorithm, MISHELF microscopy is capable of high-resolution (micron range) phase-retrieved (twin image elimination) biological imaging of dynamic events. In this contribution, MISHELF microscopy is demonstrated through qualitative concept description, algorithm implementation, and experimental validation using both a synthetic object (resolution test target) and a biological sample (swine sperm sample) for the case of three (RGB) illumination wavelengths. The proposed method becomes in an alternative instrument improving the capabilities of existing lensless microscopes.

Fast Label-Free Cytoskeletal Network Imaging in Living Mammalian Cells

We present a full-field technique that allows label-free cytoskeletal network imaging inside living cells. This noninvasive technique allows monitoring of the cytoskeleton dynamics as well as interactions between the latter and organelles on any timescale. It is based on high-resolution quantitative phase imaging (modified Quadriwave lateral shearing interferometry) and can be directly implemented using any optical microscope without modification. We demonstrate the capability of our setup on fixed and living Chinese hamster ovary cells, showing the cytoskeleton dynamics in lamellipodia during protrusion and mitochondria displacement along the cytoskeletal network. In addition, using the quantitative function of the technique, along with simulation tools, we determined the refractive index of a single tubulin microtubule to be ntubu=2.36±0.6 at λ=527 nm.

Nanoscale quantitative phase imaging using XOR-based X-ray differential interference contrast microscopy

Abstract We propose a quantitative x-ray phase imaging technique using previously demonstrated XOR objectives. The fabrication process of XOR is identical to that of a Fresnel zone-plate and as a result the same finest outermost zone width, thus spatial resolution, can be achieved. A series of phase shifts from 0 to 2 π is implemented by shifting the relative position of the grating with respect to the zone-plate. Both qualitative differential interference contrast imaging and quantitative phase reconstruction are demonstrated. We expect this high-resolution quantitative x-ray phase imaging capability to further enhance the utility of existing x-ray microscopy facilities.

Harmonically matched grating-based full-field quantitative high-resolution phase microscope for observing dynamics of transparent biological samples

We have developed a full-field high resolution quantitative phase imaging technique for observing dynamics of transparent biological samples. By using a harmonically matched diffraction grating pair (600 and 1200 lines/mm), we were able to obtain non-trivial phase difference (other than 0 degrees or 180 degrees) between the output ports of the gratings. Improving upon our previous design, our current system mitigates astigmatism artifacts and is capable of high resolution imaging. This system also employs an improved phase extraction algorithm. The system has a lateral resolution of 1.6 mum and a phase sensitivity of 62 mrad. We employed the system to acquire high resolution phase images of onion skin cells and a phase movie of amoeba proteus in motion.