Laboratory phase-contrast nanotomography of unstained Bombus terrestris compound eyes.

Imaging the visual systems of bumblebees and other pollinating insects may increase understanding of their dependence on specific habitats and how they will be affected by climate change. Current high-resolution imaging methods are either limited to two dimensions (light- and electron microscopy) or have limited access (synchrotron radiation x-ray tomography). For x-ray imaging, heavy metal stains are often used to increase contrast. Here, we present micron-resolution imaging of compound eyes of buff-tailed bumblebees (Bombus terrestris) using a table-top x-ray nanotomography (nano-CT) system. By propagation-based phase-contrast imaging, the use of stains was avoided and the microanatomy could more accurately be reconstructed than in samples stained with phosphotungstic acid or osmium tetroxide. The findings in the nano-CT images of the compound eye were confirmed by comparisons with light- and transmission electron microscopy of the same sample and finally, comparisons to synchrotron radiation tomography as well as to a commercial micro-CT system were done.

High sensitivity X-ray phase contrast imaging by laboratory grating-based interferometry at high Talbot order geometry.

X-ray phase contrast imaging is a powerful analysis technique for materials science and biomedicine. Here, we report on laboratory grating-based X-ray interferometry employing a microfocus X-ray source and a high Talbot order (35th) asymmetric geometry to achieve high angular sensitivity and high spatial resolution X-ray phase contrast imaging in a compact system (total length <1 m). The detection of very small refractive angles (∼50 nrad) at an interferometer design energy of 19 keV was enabled by combining small period X-ray gratings (1.0, 1.5 and 3.0 µm) and a single-photon counting X-ray detector (75 µm pixel size). The performance of the X-ray interferometer was fully characterized in terms of angular sensitivity and spatial resolution. Finally, the potential of laboratory X-ray phase contrast for biomedical imaging is demonstrated by obtaining high resolution X-ray phase tomographies of a mouse embryo embedded in solid paraffin and a formalin-fixed full-thickness sample of human left ventricle in water with a spatial resolution of 21.5 µm.

Phase-sensitive, angle-resolved light-scattering microscopy of single cells.

We report what is to our knowledge the first use of Fourier phase microscopy (FPM) to estimate diameters of individual single-micrometer beads and to classify cells based upon changes in scatterer size distribution. FPM, a quantitative phase imaging (QPI) method, combines the planar illumination typically used in off-axis QPI (ideal for Mie theory analysis) with the common-path geometry typically used in on-axis QPI (ideal for optimizing angular scattering range). Low-spatial-frequency imaging artifacts inherent to FPM have negligible impact upon these angular-domain applications. The system is simple to align and stable, and requires no external reference beam. Angular scattering patterns obtained from single 1 µm polystyrene beads in glycerol (Δn=0.11) display unprecedented fidelity to Mie theory, produce diameter estimates consistent with the manufacturer's specifications, and offer precision on the scale of tens of nanometers. Measurements of macrophages at different stages of antibody-dependent cellular phagocytosis demonstrate the ability to detect changes in a cell's scattering caused by the presence of phagocytosed material within.

Zernike phase-contrast full-field transmission X-ray nanotomography for 400 micrometre-sized samples.

Full-field X-ray nanotomography based on a Fresnel zone plate offers a promising and intuitive approach to acquire high-quality phase-contrast images with a spatial resolution of tens of nanometres, and is applicable to both synchrotron radiation and laboratory sources. However, its small field of view (FOV) of tens of micrometres provides limited volume information, which primarily limits its application fields. This work proposes a method for expanding the FOV as the diameter of the objective zone plate, which provides a 400 µm FOV at below 500 nm resolution with Zernike phase contrast. General applications of large-volume nanotomography are demonstrated in integrated circuit microchips and Artemia cysts. This method can be useful for imaging/analyzing industrial and biological samples where bulk properties are important or the sample is difficult to section.

3D virtual histology of human pancreatic tissue by multiscale phase-contrast X-ray tomography.

A multiscale three-dimensional (3D) virtual histology approach is presented, based on two configurations of propagation phase-contrast X-ray tomography, which have been implemented in close proximity at the GINIX endstation at the beamline P10/PETRA III (DESY, Hamburg, Germany). This enables the 3D reconstruction of characteristic morphological features of human pancreatic normal and tumor tissue, as obtained from cancer surgery, first in the form of a large-scale overview by parallel-beam illumination, followed by a zoom into a region-of-interest based on zoom tomography using a Kirkpatrick-Baez mirror with additional waveguide optics. To this end 1 mm punch biopsies of the tissue were taken. In the parallel tomography, a volumetric throughput on the order of 0.01 mm3 s-1 was achieved, while maintaining the ability to segment isolated cells. With a continuous rotation during the scan, a total acquisition time of less than 2 min was required for a full tomographic scan. Using the combination of both setups, islets of Langerhans, a three-dimensional cluster of cells in the endocrine part of the pancreas, could be located. Cells in such an islet were segmented and visualized in 3D. Further, morphological alterations of tumorous tissue of the pancreas were characterized. To this end, the anisotropy parameter Ω, based on intensity gradients, was used in order to quantify the presence of collagen fibers within the entire biopsy specimen. This proof-of-concept experiment of the multiscale approach on human pancreatic tissue paves the way for future 3D virtual pathology.

Scratch assay microscopy: A reaction-diffusion equation approach for common instruments and data.

Scratch assay is an easy and widely used "in vitro" technique to study cell migration and proliferation. In this work we focus on its modelling and on the capability to distinguish between these two phenomena that the simpler and common models are not able to disentangle. We adapted a model based on reaction-diffusion equation for being used with common microscopy instruments/data and therefore taking place in the gap between simpler modelling approaches and complex ones. An optimized image analysis pipeline and numerical least-squares fit provide estimates of the scratch proliferation and diffusion coefficients l and D. This work is intended as a first of a series in which the model is tested and its robustness and reproducibility are evaluated. Test samples were NIH3T3 cells scratch assays with proliferation and migration stimulated by varying the foetal bovine serum amount in the culture medium (10%, 7.5%, 5% and 2.5%). Results demonstrate, notwithstanding an expected l-D anticorrelation, the model capability to disentangle them. The 7.5% serum treatment can be identified as the model sensitivity limit. Treat-control l and D variations showed an intra-experiment reproducibility (∼±0.05∕h and ∼±200µm2∕h respectively) consistent with single fit typical uncertainties (∼±0.02∕h and ∼±300µm2∕h respectively).

Polarization grating based on diffraction phase microscopy for quantitative phase imaging of paramecia.

This study presents a polarization grating based diffraction phase microscopy (PG-DPM) and its application in bio-imaging. Compared with traditional diffraction phase microscopy (DPM) of which the fringe contrast is sample-dependent, the fringe contrast of PG-DPM is adjustable by changing the polarization of the illumination beam. Moreover, PG-DPM has been applied to real-time phase imaging of live paramecia for the first time. The study reveals that paramecium has self-helical forward motion characteristics, or more specifically, 77% clockwise and 23% anti-clockwise rotation when moving forward. We can envisage that PG-DPM will be applied to many different fields.

Photonic Spin-Multiplexing Metasurface for Switchable Spiral Phase Contrast Imaging.

As the two most representative operation modes in an optical imaging system, bright-field imaging and phase contrast imaging can extract different morphological information on an object. Developing a miniature and low-cost system capable of switching between these two imaging modes is thus very attractive for a number of applications, such as biomedical imaging. Here, we propose and demonstrate that a Fourier transform setup incorporating an all-dielectric metasurface can perform a two-dimensional spatial differentiation operation and thus achieve isotropic edge detection. In addition, the metasurface can provide two spin-dependent, uncorrelated phase profiles across the entire visible spectrum. Therefore, based on the spin-state of incident light, the system can be used for either diffraction-limited bright-field imaging or isotropic edge-enhanced phase contrast imaging. Combined with the advantages of planar architecture and ultrathin thickness of the metasurface, we envision this approach may open new vistas in the very interdisciplinary field of imaging and microscopy.

Analysis of Cell Division Frequency in the Root Apical Meristem of Lycophytes, Non-seed Vascular Plants, Using EdU Labeling.

The organization of the root apical meristem (RAM) provides insights into the evolution of roots in vascular plants. The RAM of seed plants has a quiescent center (QC), in which the cells divide infrequently and function to maintain neighboring stem cells. However, the existence of a QC and the mechanisms of RAM maintenance in non-seed plants are poorly understood. We analyzed the RAM organization of lycophytes focusing on cell division activity using the EdU labeling method and showed that the RAM of Lycopodium species has a region with a very low cell division frequency, which was named the QC-like region. Here, we describe an in situ EdU labeling method for the RAM of growing roots in nature.

Digital Holographic Imaging as a Method for Quantitative, Live Cell Imaging of Drug Response to Novel Targeted Cancer Therapies.

Digital holographic imaging (DHI) is a noninvasive, live cell imaging technique that enables long-term quantitative visualization of cells in culture. DHI uses phase-shift imaging to monitor and quantify cellular events such as cell division, cell death, cell migration, and drug responses. In recent years, the application of DHI has expanded from its use in the laboratory to the clinical setting, and currently it is being developed for use in theranostics. Here, we describe the use of the DHI platform HoloMonitorM4 to evaluate the effects of novel, targeted cancer therapies on cell viability and proliferation using the HeLa cancer cell line as a model. We present single cell tracking and population-wide analysis of multiple cell morphology parameters.

Scatter Enhanced Phase Contrast Microscopy for Discriminating Mechanisms of Active Nanoparticle Transport in Living Cells.

Understanding the uptake and transport dynamics of engineered nanomaterials (ENMs) by mammalian cells is an important step in designing next-generation drug delivery systems. However, to track these materials and their cellular interactions, current studies often depend on surface-bound fluorescent labels, which have the potential to alter native cellular recognition events. As a result, there is still a need to develop methods capable of monitoring ENM-cell interactions independent of surface modification. Addressing these concerns, here we show how scatter enhanced phase contrast (SEPC) microscopy can be extended to work as a generalized label-free approach for monitoring nanoparticle uptake and transport dynamics. To determine which materials can be studied using SEPC, we turn to Lorenz-Mie theory, which predicts that individual particles down to ∼35 nm can be observed. We confirm this experimentally, demonstrating that SEPC works for a variety of metal and metal oxides, including Au, Ag, TiO2, CeO2, Al2O3, and Fe2O3 nanoparticles. We then demonstrate that SEPC microscopy can be used in a quantitative, time-dependent fashion to discriminate between distinct modes of active cellular transport, including intracellular transport and membrane-assisted transport. Finally, we combine this technique with microcontact printing to normalize transport dynamics across multiple cells, allowing for a careful study of ensemble TiO2 nanoparticle uptake. This revealed three distinct regions of particle transport across the cell, indicating that membrane dynamics play an important role in regulating particle flow. By avoiding fluorescent labels, SEPC allows for a rational exploration of the surface properties of nanomaterials in their native state and their role in endocytosis and cellular transport.

One-shot phase-recovery using a cellphone RGB camera on a Jamin-Lebedeff microscope.

Jamin-Lebedeff (JL) polarization interference microscopy is a classical method for determining the change in the optical path of transparent tissues. Whilst a differential interference contrast (DIC) microscopy interferes an image with itself shifted by half a point spread function, the shear between the object and reference image in a JL-microscope is about half the field of view. The optical path difference (OPD) between the sample and reference region (assumed to be empty) is encoded into a color by white-light interference. From a color-table, the Michel-Levy chart, the OPD can be deduced. In cytology JL-imaging can be used as a way to determine the OPD which closely corresponds to the dry mass per area of cells in a single image. Like in other interference microscopy methods (e.g. holography), we present a phase retrieval method relying on single-shot measurements only, thus allowing real-time quantitative phase measurements. This is achieved by adding several customized 3D-printed parts (e.g. rotational polarization-filter holders) and a modern cellphone with an RGB-camera to the Jamin-Lebedeff setup, thus bringing an old microscope back to life. The algorithm is calibrated using a reference image of a known phase object (e.g. optical fiber). A gradient-descent based inverse problem generates an inverse look-up-table (LUT) which is used to convert the measured RGB signal of a phase-sample into an OPD. To account for possible ambiguities in the phase-map or phase-unwrapping artifacts we introduce a total-variation based regularization. We present results from fixed and living biological samples as well as reference samples for comparison.

New reconstruction method for few-view grating-based phase-contrast imaging via dictionary learning.

Grating-based phase-contrast is a hot topic in recent years owing to its excellent imaging contrast capability on soft tissues. Although it is compatible with conventional X-ray tubes and applicable in many fields, long scanning time, and high radiation dose obstruct its wider use in clinical and medical fields, especially for computed tomography applications. In this study, we solve this challenge by reducing the projection views and compensating the loss of reconstruction quality through dual-dictionary learning algorithm. The algorithm is implemented in two steps. First, estimated high-quality absorption images are obtained from the first dual-quality dictionary learning, which uses the correspondence between high-quality images and low-quality ones reconstructed from highly under-sampled data. Then, the second absorption-phase dual-modality dictionary learning is adopted to yield both estimated phase and absorption images, resulting in complementary information for both modality images. Afterwards the absorption and phase images are gradually improved in iterative reconstructions. By using SSIM RMSE measurements and visual assessment for enlarged regions of interest, our proposed method can improve the resolution of these two modality images and recover smaller structures, as compared to conventional methods.

Mobile-phone-based Rheinberg microscope with a light-emitting diode array.

Mobile phone technology has led to implementation of portable and inexpensive microscopes. Light-emitting diode (LED) array microscopes support various multicontrast imaging by flexible illumination patterns of the LED array that can be achieved without changing the optical components of the microscope. Here, we demonstrate a mobile-phone-based LED array microscope to realize multimodal imaging with bright-field, dark-field, differential phase-contrast, and Rheinberg illuminations using as few as 37 LED bulbs. Using this microscope, we obtained high-contrast images of living cells. Furthermore, by changing the color combinations of Rheinberg illumination, we were able to obtain images of living chromatic structures with enhanced or diminished contrast. This technique is expected to be a foundation for high-contrast microscopy used in modern field studies.

Multi-modal chip-based fluorescence and quantitative phase microscopy for studying inflammation in macrophages.

Total internal reflection fluorescence (TIRF) microscopy benefits from high-sensitivity, low background noise, low photo-toxicity and high-contrast imaging of sub-cellular structures close to the membrane surface. Although, TIRF microscopy provides high-contrast imaging it does not provide quantitative information about morphological features of the biological cells. Here, we propose an integrated waveguide chip-based TIRF microscopy and label-free quantitative phase imaging (QPI). The evanescent field present on top of a waveguide surface is used to excite the fluorescence and an upright microscope is used to collect the signal. The upright microscope is converted into a Linnik-type interferometer to sequentially extract both the quantitative phase information and TIRF images of the cells. Waveguide chip-based TIRF microscopy benefits from decoupling of illumination and collection light path, large field of view imaging and pre-aligned configuration for multi-color TIRF imaging. The proposed multi-modal microscopy is used to study inflammation caused by lipopolysaccharide (LPS) on rat macrophages. The TIRF microscopy showed that LPS inflammatory molecule disrupts the cell membrane and causes cells to significantly expand across a substrate. While, QPI module quantified changes in the sub-cellular content of the LPS challenged macrophages, showing a net decrease in its maximum phase values.

Endoscopic diffraction phase microscopy.

In this Letter, we present, to our knowledge, the first endoscopic diffraction phase microscopy (eDPM) system. This instrument consists of a gradient-index-lens-based endoscope probe followed by a DPM module, which enables single-shot phase imaging at a single-cell-level resolution. Using the phase information provided by eDPM, we show that the geometric aberrations associated with the endoscope can be reduced by digitally applying a spectral phase filter to the raw data. The filter function is a linear combination of polynomials with weighting optimized to improve resolution. We validate the principle of the proposed method using reflective semiconductor samples and blood cells. This research extends the current scope of quantitative phase imaging applications, and proves its potential for future in vivo studies.

GPU-based deep convolutional neural network for tomographic phase microscopy with ℓ1 fitting and regularization.

Tomographic phase microscopy (TPM) is a unique imaging modality to measure the three-dimensional refractive index distribution of transparent and semitransparent samples. However, the requirement of the dense sampling in a large range of incident angles restricts its temporal resolution and prevents its application in dynamic scenes. Here, we propose a graphics processing unit-based implementation of a deep convolutional neural network to improve the performance of phase tomography, especially with much fewer incident angles. As a loss function for the regularized TPM, the ℓ1-norm sparsity constraint is introduced for both data-fidelity term and gradient-domain regularizer in the multislice beam propagation model. We compare our method with several state-of-the-art algorithms and obtain at least 14 dB improvement in signal-to-noise ratio. Experimental results on HeLa cells are also shown with different levels of data reduction.

Isotropic differential phase contrast microscopy for quantitative phase bio-imaging.

Quantitative phase imaging (QPI) has been investigated to retrieve optical phase information of an object and applied to biological microscopy and related medical studies. In recent examples, differential phase contrast (DPC) microscopy can recover phase image of thin sample under multi-axis intensity measurements in wide-field scheme. Unlike conventional DPC, based on theoretical approach under partially coherent condition, we propose a new method to achieve isotropic differential phase contrast (iDPC) with high accuracy and stability for phase recovery in simple and high-speed fashion. The iDPC is simply implemented with a partially coherent microscopy and a programmable thin-film transistor (TFT) shield to digitally modulate structured illumination patterns for QPI. In this article, simulation results show consistency of our theoretical approach for iDPC under partial coherence. In addition, we further demonstrate experiments of quantitative phase images of a standard micro-lens array, as well as label-free live human cell samples.

Using machine-learning to optimize phase contrast in a low-cost cellphone microscope.

Cellphones equipped with high-quality cameras and powerful CPUs as well as GPUs are widespread. This opens new prospects to use such existing computational and imaging resources to perform medical diagnosis in developing countries at a very low cost. Many relevant samples, like biological cells or waterborn parasites, are almost fully transparent. As they do not exhibit absorption, but alter the light's phase only, they are almost invisible in brightfield microscopy. Expensive equipment and procedures for microscopic contrasting or sample staining often are not available. Dedicated illumination approaches, tailored to the sample under investigation help to boost the contrast. This is achieved by a programmable illumination source, which also allows to measure the phase gradient using the differential phase contrast (DPC) [1, 2] or even the quantitative phase using the derived qDPC approach [3]. By applying machine-learning techniques, such as a convolutional neural network (CNN), it is possible to learn a relationship between samples to be examined and its optimal light source shapes, in order to increase e.g. phase contrast, from a given dataset to enable real-time applications. For the experimental setup, we developed a 3D-printed smartphone microscope for less than 100 $ using off-the-shelf components only such as a low-cost video projector. The fully automated system assures true Koehler illumination with an LCD as the condenser aperture and a reversed smartphone lens as the microscope objective. We show that the effect of a varied light source shape, using the pre-trained CNN, does not only improve the phase contrast, but also the impression of an improvement in optical resolution without adding any special optics, as demonstrated by measurements.

Improved contrast microscopy: modification of bright field for urine sediment visualisation.

BACKGROUND: Phase contrast microscopy is the recommended technique for urine sediment examination. Bright field microscopy does not differentiate objects with slight changes in the refractive index and hence phase contrast is a superior alternative. METHODS: In this article, we describe a novel method to improve contrast in bright field microscopy. A strategically placed disc of specific dimensions enhances the diffraction of rays by Fresnel principle causing a shift in wavelength in the rays which are perceived as differences in contrast by the eye due to constructive and destructive interference. RESULTS: Epithelial cells, red blood cells (RBCs), dysmorphic red blood cells, casts, bacteria and crystals are easily seen and differentiated by this technique. CONCLUSIONS: The images obtained are similar to those obtained by phase contrast microscopy.