Optical Diffraction Tomography Technique Research Articles

Transport-of-intensity Fourier ptychographic diffraction tomography: defying the matched illumination condition

Optical diffraction tomography (ODT) is a promising label-free three-dimensional (3D) microscopic method capable of measuring the 3D refractive index (RI) distribution of optically transparent samples (e.g., unlabeled biological cells). In recent years, non-interferometric ODT techniques have received increasing attention for their system simplicity, speckle-free imaging quality, and compatibility with existing microscopes. However, ODT methods for implementing non-interferometric measurements in high numerical aperture (NA) microscopy systems are often plagued by low-frequency missing problems—a consequence of violating the matched illumination condition. Here, we present transport-of-intensity Fourier ptychographic diffraction tomography (TI-FPDT) to address this challenging issue by combining ptychographic angular diversity with additional “transport of intensity” measurements. TI-FPDT exploits the defocused phase contrast to circumvent the stringent requirement on the illumination NA imposed by the matched illumination condition. It effectively overcomes the reconstruction quality deterioration and RI underestimation problems in conventional FPDT, as demonstrated by high-resolution tomographic imaging of various unlabeled transparent samples (including microspheres, USAF targets, HeLa cells, and C2C12 cells). Due to its simplicity and effectiveness, TI-FPDT is anticipated to open new possibilities for label-free 3D microscopy in various biomedical applications.

Characterization of two-photon photopolymerization fabrication using high-speed optical diffraction tomography

Two-photon photopolymerization (TPP) has recently become a popular method for the fabrication of three-dimensional (3D) micro- and nanostructures. The reproduction fidelity of the designed micro- and nanostructures is influenced by experimental writing conditions, including laser power, exposure time, etc. To determine the appropriate writing parameters, characterization of morphological features and surface roughness during the experiment is needed. Traditional characterization methods for TPP, e.g., scanning electron microscopy and atomic force microscopy, have limited speed and cannot study internal structures without invasive approaches. Optical diffraction tomography (ODT) is an emerging label-free 3D imaging technique based on reconstructing the object’s 3D refractive index (RI) distribution with diffraction-limited resolution. Here, we propose a non-invasive solution to fully characterize the TPP-fabricated structures using a high-speed ODT technique, which can eliminate the need for complex sample preparation, such as fluorescence labelling or metal-coating, and achieve a full 3D measurement time of 6 ms. By visualizing and studying different TPP-fabricated structures, including embedded spirals and cubes, via the ODT system, the fabrication quality, including 3D morphological features, exposure levels, and surface roughness, can be examined quantitatively. The results suggest our method can effectively improve the fabrication quality and reproducibility of TPP, generating impacts on the nanofabrication community.

Ring stage classification of Babesia microti and Plasmodium falciparum using optical diffraction 3D tomographic technique

BackgroundBabesia is an intraerythrocytic parasite often misdiagnosed as a malaria parasite, leading to inappropriate treatment of the disease especially in co-endemic areas. In recent years, optical diffraction tomography (ODT) has shown great potential in the field of pathogen detection by quantification of three-dimensional (3D) imaging tomograms. The 3D imaging of biological cells is crucial to investigate and provide valuable information about the mechanisms behind the pathophysiology of cells and tissues.MethodsThe early ring stage of P. falciparum were obtained from stored stock of infected RBCs and of B. microti were obtained from infected patients during diagnosis. The ODT technique was applied to analyze and characterize detailed differences between P. falciparum and B. microti ring stage at the single cell level. Based on 3D quantitative information, accurate measurement was performed of morphological, biochemical, and biophysical parameters.ResultsAccurate measurements of morphological parameters indicated that the host cell surface area at the ring stage in B. microti was significantly smaller (140.2 ± 17.1 µm2) than that in P. falciparum (159.0 ± 15.2 µm2), and sphericities showed higher levels in B. microti-parasitized cells (0.66 ± 0.05) than in P. falciparum (0.60 ± 0.04). Based on biochemical parameters, host cell hemoglobin level was significantly higher and membrane fluctuations were respectively more active in P. falciparum-infected cells (30.25 ± 2.96 pg; 141.3 ± 24.68 nm) than in B. microti (27.28 ± 3.52 pg; 110.1 ± 38.83 nm). The result indicates that P. falciparum more actively altered host RBCs than B. microti.ConclusionAlthough P. falciparum and B. microti often show confusable characteristics under the microscope, and the actual three-dimensional properties are different. These differences could be used in differential clinical diagnosis of erythrocytes infected with B. microti and P. falciparum.Graphical

The Xenopus spindle is as dense as the surrounding cytoplasm.

The mitotic spindle is a self-organizing molecular machine, where hundreds of different molecules continuously interact to maintain a dynamic steady state. While our understanding of key molecular players in spindle assembly is significant, it is still largely unknown how the spindle's material properties emerge from molecular interactions. Here, we use correlative fluorescence imaging and label-free three-dimensional optical diffraction tomography (ODT) to measure the Xenopus spindle's mass density distribution. While the spindle has been commonly referred to as a denser phase of the cytoplasm, we find that it has the same density as its surrounding, which makes it neutrally buoyant. Molecular perturbations suggest that spindle mass density can be modulated by tuning microtubule nucleation and dynamics. Together, ODT provides direct, unbiased, and quantitative information of the spindle's emergent physical properties-essential to advance predictive frameworks of spindle assembly and function.

Low-coherence optical diffraction tomography using a ferroelectric liquid crystal spatial light modulator.

Optical diffraction tomography (ODT) is a three-dimensional (3D) label-free imaging technique. The 3D refractive index distribution of a sample can be reconstructed from multiple two-dimensional optical field images via ODT. Herein, we introduce a temporally low-coherence ODT technique using a ferroelectric liquid crystal spatial light modulator (FLC SLM). The fast binary-phase modulation provided by the FLC SLM ensures the high spatiotemporal resolution. To reduce coherent noise, a superluminescent light-emitting diode is used as an economic low-coherence light source. We demonstrate the performance of the proposed system using various samples, including colloidal microspheres and live epithelial cells.

Label-free bioanalysis of Leishmania infantum using refractive index tomography with partially coherent illumination.

In this work, we report the use of refractive index (RI) tomography for quantitative analysis of unstained DH82 cell line infected with Leishmania infantum. The cell RI is reconstructed by using a modality of optical diffraction tomography technique that employs partially coherent illumination, thus enabling inherent compatibility with conventional wide-field microscopes. The experimental results demonstrate that the cell dry mass concentration (DMC) obtained from the RI allows for reliable detection and quantitative characterization of the infection and its temporal evolution. The RI provides important insight for studying morphological changes, particularly membrane blebbing linked to an apoptosis (cell death) process induced by the disease. Moreover, the results evidence that infected DH82 cells exhibit a higher DMC than healthy samples. These findings open up promising perspectives for clinical diagnosis of Leishmania.

Label-free quantitative 3D tomographic imaging for partially coherent light microscopy.

Crucial benefits provided by partially coherent light microscopy such as improved spatial resolution, optical sectioning and speckle-noise suppression are exploited here to achieve 3D quantitative imaging: reconstruction of the object refractive index (RI). We present a partially coherent optical diffraction tomography technique (PC-ODT) that can be easily implemented in commercially available bright-field microscopes. We show that the high numerical apertures of the objective and condenser lenses, together with optical refocusing, are main issues for achieving fast and successful 3D RI reconstruction of weak objects. In particular, the optical refocusing is performed by a high-speed focus tunable lens mounted in front of the digital camera enabling compatibility with commercial microscopes. The technique is experimentally demonstrated on different examples: diatom cells (biosilica shells), polystyrene micro-spheres and blood cells. The results confirm the straightforward 3D-RI reconstruction of the samples providing valuable quantitative information for their analysis. Thus, the PC-ODT can be considered as an efficient and affordable alternative to coherent ODT which requires specially designed holographic microscopes.

Optical diffraction tomography microscopy with transport of intensity equation using a light-emitting diode array

Optical diffraction tomography (ODT) is an effective label-free technique for quantitatively refractive index imaging, which enables long-term monitoring of the internal three-dimensional (3D) structures and molecular composition of biological cells with minimal perturbation. However, existing optical tomographic methods generally rely on interferometric configuration for phase measurement and sophisticated mechanical systems for sample rotation or beam scanning. Thereby, the measurement is suspect to phase error coming from the coherent speckle, environmental vibrations, and mechanical error during data acquisition process. To overcome these limitations, we present a new ODT technique based on non-interferometric phase retrieval and programmable illumination emitting from a light-emitting diode (LED) array. The experimental system is built based on a traditional bright field microscope, with the light source replaced by a programmable LED array, which provides angle-variable quasi-monochromatic illumination with an angular coverage of ±37 degrees in both x and y directions (corresponding to an illumination numerical aperture of ∼0.6). Transport of intensity equation (TIE) is utilized to recover the phase at different illumination angles, and the refractive index distribution is reconstructed based on the ODT framework under first Rytov approximation. The missing-cone problem in ODT is addressed by using the iterative non-negative constraint algorithm, and the misalignment of the LED array is further numerically corrected to improve the accuracy of refractive index quantification. Experiments on polystyrene beads and thick biological specimens show that the proposed approach allows accurate refractive index reconstruction while greatly reduced the system complexity and environmental sensitivity compared to conventional interferometric ODT approaches.

Optical diffraction tomography techniques for the study of cell pathophysiology

Three-dimensional imaging of biological cells is crucial for the investigation of cell biology, providing valuable information to reveal the mechanisms behind pathophysiology of cells and tissues. Recent advances in optical diffraction tomography (ODT) have demonstrated the potential for the study of various cells with its unique advantages of quantitative and label-free imaging capability. To provide insight on this rapidly growing field of research and to discuss its applications in biology and medicine, we present the summary of the ODT principle and highlight recent studies utilizing ODT with the emphasis on the applications to the pathophysiology of cells.

Real-time visualization of 3-D dynamic microscopic objects using optical diffraction tomography

3-D refractive index (RI) distribution is an intrinsic bio-marker for the chemical and structural information about biological cells. Here we develop an optical diffraction tomography technique for the real-time reconstruction of 3-D RI distribution, employing sparse angle illumination and a graphic processing unit (GPU) implementation. The execution time for the tomographic reconstruction is 0.21 s for 96(3) voxels, which is 17 times faster than that of a conventional approach. We demonstrated the real-time visualization capability with imaging the dynamics of Brownian motion of an anisotropic colloidal dimer and the dynamic shape change in a red blood cell upon shear flow.

Optical diffraction tomography within a variational Bayesian framework

Optical diffraction tomography techniques aim to find an image of an unknown object (e.g. a map of its refraction index) using measurements of the scattered field that results from its interaction with a known interrogating wave. We address this issue as a nonlinear inverse scattering problem. The forward model is based upon domain integral representations of the electric field whose discrete counterparts are obtained by means of a method of moments. The inverse problem is tackled in a Bayesian estimation framework involving a hierarchical prior model that accounts for the piece-wise homogeneity of the object. A joint unsupervised estimation approach is adopted to estimate the induced currents, the contrast and all the other parameters introduced in the prior model. As an analytic expression for the joint maximum a posteriori (MAP) and posterior mean (PM) estimators is hard to obtain, a tractable approximation of the latter is proposed. This approximation is based upon a variational Bayesian technique and consists in the best separable distribution that approximates the true posterior distribution in the Kullback–Leibler sense. This leads to an implicit parametric optimization scheme which is solved iteratively.

Modal-based tomographic imaging from far-zone observations: multifrequency case

A recently developed optical diffraction tomography technique [J. Opt. Soc. Am. A 26, 19 (2009)] is modified to handle the special case of a dispersionless scatterer, where the relative permittivity contrast of the object is independent of the operation frequency, allowing the fusion of multifrequency data. The modified technique is illustrated with a simulation scenario involving a dispersionless and lossless object being probed with plane waves of distinct incidence angles and frequencies. Superiority of the fusion of multifrequency data over the use of single-frequency data is demonstrated.

Modal-based tomographic imaging from far-zone observations

A novel method of optical diffraction tomography (ODT) to image weakly scattering, electrically large, two-dimensional (2D) objects using the far-zone scattered field data is presented. The proposed technique is based on the expansion of the target object function in terms of Fourier-Bessel basis functions and an alternative approximation for the total electric field within the support of the investigated scatterer. Analytical (Mie) plane-wave scattering by a layered, circularly symmetric, lossy cylinder, and finite-difference time-domain simulations involving plane-wave scattering by a more general, lossless phantom are utilized to compare the performance of the proposed method with that of the standard ODT techniques, which are based on the Born approximation and the Fourier diffraction theorem. Quantitative and qualitative superiority of the presented method is demonstrated. The proposed 2D technique can be readily extended to more realistic three-dimensional cases. With proper (cylindrical-spherical) receiver configuration, the proposed method can be used without being confined to far-zone observations.

Living specimen tomography by digital holographic microscopy: morphometry of testate amoeba

This paper presents an optical diffraction tomography technique based on digital holographic microscopy. Quantitative 2-dimensional phase images are acquired for regularly-spaced angular positions of the specimen covering a total angle of pi, allowing to built 3-dimensional quantitative refractive index distributions by an inverse Radon transform. A 20x magnification allows a resolution better than 3 microm in all three dimensions, with accuracy better than 0.01 for the refractive index measurements. This technique is for the first time to our knowledge applied to living specimen (testate amoeba, Protista). Morphometric measurements are extracted from the tomographic reconstructions, showing that the commonly used method for testate amoeba biovolume evaluation leads to systematic under evaluations by about 50%.