Quantitative Phase Information Research Articles

Investigating fibroblast cells under "safe" and "injurious" blue-light exposure by holographic microscopy.

The exposure to visible light has been shown to exert various biological effects, such as erythema and retinal degeneration. However, the phototoxicity mechanisms in living cells are still not well understood. Here we report a study on the temporal evolution of cell morphology and volume during blue light exposure. Blue laser irradiation is switched during the operation of a digital holography (DH) microscope between what we call here "safe" and "injurious" exposure (SE & IE). The results reveal a behaviour that is typical of necrotic cells, with early swelling and successive leakage of the intracellular liquids when the laser is set in the "injurious" operation. In the phototoxicity investigation reported here the light dose modulation is performed through the very same laser light source adopted for monitoring the cell's behaviour by digital holographic microscope. We believe the approach may open the route to a deep investigation of light-cell interactions, with information about death pathways and threshold conditions between healthy and damaged cells when subjected to light-exposure. 3D Morphology and quantitative phase information from late stage of necrosis cell death.

Spatial heterodyne scanning laser confocal holographic microscopy

Scanning laser confocal holographic microscopy using a spatial heterodyne detection method is presented. Spatial heterodyne detection technique employs a Mach-Zehnder interferometer with the reference beam frequency shifted by two acousto-optic modulators (AOM) relative to the object beam frequency. Different from the traditional temporal heterodyne detection technique in which hundreds temporal samples are taken at each scanning point to achieve the complex signal, the spatial heterodyne detection technique generates spatial interference fringes by use of a linear tempo-spatial relation provided by galvanometer scanning in a typical line-scanning confocal microscope or for the slow-scanning on one dimension in a point-scanning confocal microscope, thereby significantly reducing sampling rate and increasing the signal to noise ratio under the same illumination compared to the traditional temporal heterodyne counterpart. The proposed spatial heterodyne detection scheme applies to both line-scanning and point-scanning confocal microscopes. In this paper, we present the mathematical principles of the spatial heterodyne detection method and experimental schemes for both line-scanning and point-scanning confocal microscopes. Computer experiments are provided to demonstrate the validity of this idea. The presented spatial heterodyne scanning laser confocal holographic microscope (SH-SLCHM) can obtain both amplitude and quantitative phase information of the object field without a significant increase in complexity of optical and electronic design on the basis of a traditional scanning laser confocal microscope (SLCM). SH-SLCHM may find applications in optical metrology, in-vivo tissue imaging, and ophthalmic imaging.

High contrast 3D imaging of surfaces near the wavelength limit using tabletop EUV ptychography

Scanning electron microscopy and atomic force microscopy are well-established techniques for imaging surfaces with nanometer resolution. Here we demonstrate a complementary and powerful approach based on tabletop extreme-ultraviolet ptychography that enables quantitative full field imaging with higher contrast than other techniques, and with compositional and topographical information. Using a high numerical aperture reflection-mode microscope illuminated by a tabletop 30nm high harmonic source, we retrieve high quality, high contrast, full field images with 40nm by 80nm lateral resolution (≈1.3λ), with a total exposure time of less than 1min. Finally, quantitative phase information enables surface profilometry with ultra-high, 6Å axial resolution. In the future, this work will enable dynamic imaging of functioning nanosystems with unprecedented combined spatial (<10nm) and temporal (<10fs) resolution, in thick opaque samples, with elemental, chemical and magnetic sensitivity.

Spatiotemporal characterization of a fibrin clot using quantitative phase imaging.

Studying the dynamics of fibrin clot formation and its morphology is an important problem in biology and has significant impact for several scientific and clinical applications. We present a label-free technique based on quantitative phase imaging to address this problem. Using quantitative phase information, we characterized fibrin polymerization in real-time and present a mathematical model describing the transition from liquid to gel state. By exploiting the inherent optical sectioning capability of our instrument, we measured the three-dimensional structure of the fibrin clot. From this data, we evaluated the fractal nature of the fibrin network and extracted the fractal dimension. Our non-invasive and speckle-free approach analyzes the clotting process without the need for external contrast agents.

Imaging‐in‐Flow: Digital holographic microscopy as a novel tool to detect and classify nanoplanktonic organisms

Traditional taxonomic identification of planktonic organisms is based on light microscopy, which is both time‐ consuming and tedious. In response, novel ways of automated (machine) identification, such as flow cytometry, have been investigated over the last two decades. To improve the taxonomic resolution of particle analysis, recent developments have focused on “imaging‐ in‐ flow,” i.e., the ability to acquire microscopic images of planktonic cells in a flow‐ through mode. Imaging‐ in‐ flow systems are traditionally based on classical brightfield microscopy and are faced with a number of issues that decrease the classification performance and accuracy (e.g., projection variance of cells, migration of cells out of the focus plane). Here, we demonstrate that a combination of digital holographic microscopy (DHM) with imaging‐ in‐ flow can improve the detection and classification of planktonic organisms. In addition to light intensity information, DHM provides quantitative phase information, which generates an additional and independent set of features that can be used in classification algorithms. Moreover, the capability of digitally refocusing greatly increases the depth of field, enables a more accurate focusing of cells, and reduces the effects of position variance. Nanoplanktonic organisms similar in shape were successfully classified from images captured with an off‐ axis DHM with partial coherence. Textural features based on DHM phase information proved more efficient in separating the three tested phytoplankton species compared with shape‐ based features or textural features based on light intensity. An overall classification score of 92.4% demonstrates the potential of holographic‐ based imaging‐ in‐ flow for similar looking organisms in the nanoplankton range.

The effect of the spatial sampling rate on quantitative phase information extracted from planar and tomographic edge illumination x-ray phase contrast images

The effect of the spatial sampling rate on the quantitative phase information that can be retrieved from planar and tomographic edge illumination (EI) x-ray phase contrast imaging (XPCi) with a laboratory-based prototype scanner is analysed. The study is conducted on simulated and experimental data from a custom-built phantom. Optimal sampling rates, i.e. the minimum ones allowing the unambiguous extraction of quantitative phase measurements from the acquired data, are identified for planar and tomographic imaging. One of the key outcomes of this study is the demonstration that the optimal sampling rate in tomographic EI XPCi is low, allowing the implementation of low-dose volumetric imaging without having to compromise on quantitative accuracy.

Quantitative phase-contrast imaging through a scattering media.

We report a new technique for the recovery of quantitative phase and amplitude information of an object hidden behind a scattering medium. Two-point intensity correlation measurement together with digital holography principles are utilized for this purpose. The hologram information of the object and a reference beam is scrambled by the presence of a scattering medium in its path. A direct digital holographic recording of this scattered light does not lead to the reconstruction of actual object information. We propose the idea of recovering this hologram information from the spatially fluctuating field of a laser speckle pattern using the intensity correlation, and subsequently apply digital reconstruction of the hologram for recovery of quantitative phase and amplitude information of objects hidden by a random diffuser.

Simulation study on the quantitative phase retrieval method under two-step phase-shifting technique

Based on two-step phase-shifting technique, we present a new derivative method for phase information extraction in quantitative phase imaging (QPI). By acquiring two phase-shifted interferograms and numerically calculating their 1st order derivatives, one can directly obtain the quantitative phase information. We demonstrate the proposed method by comparing our simulated results with the experimental results of the red blood cell and the HeLa cell, respectively. It shows that our method can be generally applied to any QPI, such as on-axis and off-axis interferometry, especially for slightly off-axis interferometry, and it has the feature of efficient utility of camera spatial bandwidth and the ability of fast data processing.

A phase retrieval method of interferograms add-subtracting based on two-step phase shifting

A phase retrieval method is introduced in quantitative phase imaging (QPI) based on two-step phase-shifting technique. By acquiring two measured interferograms and calculating the addition and subtraction between them, the quantitative phase information can be directly retrieved. This method is illustrated by both theory and simulation experiments of a ball. The results of the simulation and the experiment of the red blood cell show a good agreement, demonstrating its application for studying cells.

Cell Optical Density and Molecular Composition Revealed by Simultaneous Multimodal Label-Free Imaging

We show how Raman imaging can be combined with independent but simultaneous phase measurements of unlabeled cells, with the resulting data providing information on how the light is retarded and/or scattered by molecules in the cell. We then show, for the first time to our knowledge, how the chemistry of the cell highlighted in the Raman information is related to the cell quantitative phase information revealed in digital holographic microscopy by quantifying how the two sets of spatial information are correlated. The results show that such a multimodal implementation is highly useful for the convenience of having video rate imaging of the cell during the entire Raman measurement time, allowing us to observe how the cell changes during Raman acquisition. More importantly, it also shows that the two sets of label-free data, which result from different scattering mechanisms, are complementary and can be used to interpret the composition and dynamics of the cell, where each mode supplies label-free information not available from the other mode.

Ptychography – a label free, high-contrast imaging technique for live cells using quantitative phase information

Cell imaging often relies on synthetic or genetic fluorescent labels, to provide contrast which can be far from ideal for imaging cells in their in vivo state. We report on the biological application of a, label-free, high contrast microscopy technique known as ptychography, in which the image producing step is transferred from the microscope lens to a high-speed phase retrieval algorithm. We demonstrate that this technology is appropriate for label-free imaging of adherent cells and is particularly suitable for reporting cellular changes such as mitosis, apoptosis and cell differentiation. The high contrast, artefact-free, focus-free information rich images allow dividing cells to be distinguished from non-dividing cells by a greater than two-fold increase in cell contrast, and we demonstrate this technique is suitable for downstream automated cell segmentation and analysis.

Whole-cell phase contrast imaging at the nanoscale using Fresnel Coherent Diffractive Imaging Tomography

X-ray tomography can provide structural information of whole cells in close to their native state. Radiation-induced damage, however, imposes a practical limit to image resolution, and as such, a choice between damage, image contrast, and image resolution must be made. New coherent diffractive imaging techniques, such Fresnel Coherent Diffractive Imaging (FCDI), allows quantitative phase information with exceptional dose efficiency, high contrast, and nano-scale resolution. Here we present three-dimensional quantitative images of a whole eukaryotic cell by FCDI at a spatial resolution below 70 nm with sufficient phase contrast to distinguish major cellular components. From our data, we estimate that the minimum dose required for a similar resolution is close to that predicted by the Rose criterion, considerably below accepted estimates of the maximum dose a frozen-hydrated cell can tolerate. Based on the dose efficiency, contrast, and resolution achieved, we expect this technique will find immediate applications in tomographic cellular characterisation.

Comparison of single distance phase retrieval algorithms by considering different object composition and the effect of statistical and structural noise

Phase retrieval is a technique for extracting quantitative phase information from X-ray propagation-based phase-contrast tomography (PPCT). In this paper, the performance of different single distance phase retrieval algorithms will be investigated. The algorithms are herein called phase-attenuation duality Born Algorithm (PAD-BA), phase-attenuation duality Rytov Algorithm (PAD-RA), phase-attenuation duality Modified Bronnikov Algorithm (PAD-MBA), phase-attenuation duality Paganin algorithm (PAD-PA) and phase-attenuation duality Wu Algorithm (PAD-WA), respectively. They are all based on phase-attenuation duality property and on weak absorption of the sample and they employ only a single distance PPCT data. In this paper, they are investigated via simulated noise-free PPCT data considering the fulfillment of PAD property and weakly absorbing conditions, and with experimental PPCT data of a mixture sample containing absorbing and weakly absorbing materials, and of a polymer sample considering different degrees of statistical and structural noise. The simulation shows all algorithms can quantitatively reconstruct the 3D refractive index of a quasi-homogeneous weakly absorbing object from noise-free PPCT data. When the weakly absorbing condition is violated, the PAD-RA and PAD-PA/WA obtain better result than PAD-BA and PAD-MBA that are shown in both simulation and mixture sample results. When considering the statistical noise, the contrast-to-noise ratio values decreases as the photon number is reduced. The structural noise study shows that the result is progressively corrupted by ring-like artifacts with the increase of structural noise (i.e. phantom thickness). The PAD-RA and PAD-PA/WA gain better density resolution than the PAD-BA and PAD-MBA in both statistical and structural noise study.

Three-dimensional intracellular optical coherence phase imaging

Quantitative phase imaging has many applications for label-free studies of the nanoscale structure and dynamics of cells and tissues. It has been demonstrated that optical coherence phase microscopy (OCPM) can provide quantitative phase information with very high sensitivity. The excellent phase stability of OCPM is obtained by use of a reflection from the microscope cover glass as a local reference field. For detailed intracellular studies a large numerical aperture (N.A.) objective is needed in order to obtain the required resolution. Unfortunately, this also means that the depth of field becomes too small to obtain sufficient power from the cover glass when the beam is focused into the sample. To address this issue, we designed a setup with a dual-beam sample arm. One beam with a large diameter (filling the 1.2 N.A. water immersion objective) enabled high-resolution imaging. A second beam with a small diameter (underfilling the same objective) had a larger depth of field and could detect the cover glass used as a local phase reference. The phase stability of the setup was quantified by monitoring the front and back of a cover glass. The standard deviation of the phase difference was 0.021 rad, corresponding to an optical path displacement of 0.9 nm. The lateral and axial dimensions of the confocal point spread function were 0.42 and 0.84 μm, respectively. This makes our dual-beam setup ideal for three-dimensional intracellular phase imaging.

Spectroscopic diffraction phase microscopy

We present spectroscopic diffraction phase microscopy (sDPM) as a method capable of measuring quantitative phase images at multiple wavelengths. sDPM uses a spatial light modulator at the Fourier plane of a lens to select desired wavelengths from the white light illumination of a grating. The quantitative phase information at different wavelengths allows us to decouple the refractive index and the thickness from the phase shift induced by biological cells. We demonstrate the capability of the setup by dispersion measurements of microsphere beads and RBCs.

Visualizing Escherichia coli Sub-Cellular Structure Using Sparse Deconvolution Spatial Light Interference Tomography

Studying the 3D sub-cellular structure of living cells is essential to our understanding of biological function. However, tomographic imaging of live cells is challenging mainly because they are transparent, i.e., weakly scattering structures. Therefore, this type of imaging has been implemented largely using fluorescence techniques. While confocal fluorescence imaging is a common approach to achieve sectioning, it requires fluorescence probes that are often harmful to the living specimen. On the other hand, by using the intrinsic contrast of the structures it is possible to study living cells in a non-invasive manner. One method that provides high-resolution quantitative information about nanoscale structures is a broadband interferometric technique known as Spatial Light Interference Microscopy (SLIM). In addition to rendering quantitative phase information, when combined with a high numerical aperture objective, SLIM also provides excellent depth sectioning capabilities. However, like in all linear optical systems, SLIM's resolution is limited by diffraction. Here we present a novel 3D field deconvolution algorithm that exploits the sparsity of phase images and renders images with resolution beyond the diffraction limit. We employ this label-free method, called deconvolution Spatial Light Interference Tomography (dSLIT), to visualize coiled sub-cellular structures in E. coli cells which are most likely the cytoskeletal MreB protein and the division site regulating MinCDE proteins. Previously these structures have only been observed using specialized strains and plasmids and fluorescence techniques. Our results indicate that dSLIT can be employed to study such structures in a practical and non-invasive manner.

Blood testing at the single cell level using quantitative phase and amplitude microscopy

It has recently been shown that quantitative phase imaging methods can provide clinically relevant parameters for red blood cell analysis with unprecedented detail and sensitivity. Since the quantitative phase information is dependent on both the thickness and refractive index, a major limitation to clinical translation has been a simple and practical approach to measure both simultaneously. Here we demonstrate both theoretically and experimentally that, by combining quantitative phase with a single absorption measurement, it is possible to measure both quantities at the single cell level. We validate this approach by comparing our results to those acquired using a clinical blood analyzer. This approach to decouple the thickness and refractive index for red blood cells may be used with any quantitative phase imaging method that can operate in tandem with bright field microscopy at the Soret-band wavelength.

Tissue refractive index as marker of disease

The gold standard in histopathology relies on manual investigation of stained tissue biopsies. A sensitive and quantitative method for in situ tissue specimen inspection is highly desirable, as it would allow early disease diagnosis and automatic screening. Here we demonstrate that quantitative phase imaging of entire unstained biopsies has the potential to fulfill this requirement. Our data indicates that the refractive index distribution of histopathology slides, which contains information about the molecular scale organization of tissue, reveals prostate tumors and breast calcifications. These optical maps report on subtle, nanoscale morphological properties of tissues and cells that cannot be recovered by common stains, including hematoxylin and eosin. We found that cancer progression significantly alters the tissue organization, as exhibited by consistently higher refractive index variance in prostate tumors versus normal regions. Furthermore, using the quantitative phase information, we obtained the spatially resolved scattering mean free path and anisotropy factor g for entire biopsies and demonstrated their direct correlation with tumor presence. In essence, our results show that the tissue refractive index reports on the nanoscale tissue architecture and, in principle, can be used as an intrinsic marker for cancer diagnosis.

Phase retrieval in quantitative x-ray microtomography with a single sample-to-detector distance

Phase retrieval extracts quantitative phase information from x-ray propagation-based phase-contrast images. Notwithstanding inherent approximations, phase retrieval using a single sample-to-detector distance (SDD) is very attractive, because it imposes no setup complications or additional radiation dose compared to absorption-based imaging. Considering the phase-attenuation duality (ε=δ/β, where ε is constant), a simple absorption correction factor is proposed for the modified Bronnikov algorithm in x-ray propagation-based phase-contrast computed tomography (PPCT). Moreover, a practical method for calculating the optimal ε value is proposed, which requires no prior knowledge of the sample. Tests performed on simulation and experimental data successfully distinguished different materials in a quasihomogeneous and weakly absorbing sample from a single SDD-PPCT data point.

Quantitative x-ray differential-interference-contrast microscopy with independently adjustable bias and shear

We present a quantitative x-ray phase imaging method that can be readily implemented on existing x-ray microscopy facilities. This technique utilizes Fresnel zone plates both as imaging optical elements for magnification and as second-order grating structures for phase-shifting interferometry. By making high-resolution quantitative x-ray phase information widely available, we expect this work to have significant impact on nanoscale biological and material studies.