LED based large field of view off-axis quantitative phase contrast microscopy by hologram multiplexing.

In this manuscript, we describe the development of a single shot, self-referencing wavefront division, multiplexing digital holographic microscope employing LED sources for large field of view quantitative phase imaging of biological samples. To address the difficulties arising while performing interferometry with low temporally coherent sources, an optical arrangement utilizing multiple Fresnel Biprisms is used for hologram multiplexing, enhancing the field of view and increasing the signal to noise ratio. Biprisms offers the ease of obtaining interference patterns by automatically matching the path length between the two off-axis beams. The use of low temporally coherent sources reduces the speckle noise and the cost, and the form factor of the setup. The developed technique was implemented using both visible and UV LEDs and tested on polystyrene microspheres and human erythrocytes.

Phenanthraquinone-Doped Polymethyl Methacrylate Photopolymer for Holographic Recording.

Phenanthraquinone-doped polymethyl methacrylate (PQ/PMMA) photopolymers are considered to be the most promising holographic storage media due to their unique properties, such as high stability, a simple preparation process, low price, and volumetric shrinkage. This paper reviews the development process of PQ/PMMA photopolymers from inception to the present, summarizes the process, and looks at the development potential of PQ/PMMA in practical applications.

Multispectral photoacoustic holography of elastomers from a bright background.

Photoacoustic imaging of elastomers has important biomedical value. However, a bright background, e.g., blood vessels in living tissue, brings a challenge for photoacoustic elastography. In this study, we predicted that the spectrum of photoacoustic signals from elastomers with high elasticity could appear as narrow peaks at the eigen-frequencies of elastomers, but the signals from a bright background, e.g., blood vessel, show flat broadband spectrum for their low-quality factor. Even when the two kinds of signals are mixed together, the signals from elastomers can be identified from the spectrum since they present as convex narrow peaks on a wide base. Based on this factor, we propose a multispectral photoacoustic holography to realize selective imaging of tiny elastomers. This method recovers the image only using several frequency components in photoacoustic signals, instead of the whole-band signal. Since these narrow peaks in the spectrum correspond to the eigen-vibration of elastomers, the proposed method can highlight the elastomers with high elasticity from a bright background with low elasticity. The method was validated by experiments. This study might be helpful to localize elastic anomalous areas in the tissue, such as calcification in the vascular network, microcalcification in a tumor, and implants.

Photolon Nanoporous Photoactive Material with Antibacterial Activity and Label-Free Noncontact Method for Free Radical Detection.

The worldwide increase in bacterial resistance and healthcare-associated bacterial infections pose a serious threat to human health. The antimicrobial photodynamic method reveals the opportunity for a new therapeutic approach that is based on the limited delivery of photosensitizer from the material surface. Nanoporous inorganic-organic composites were obtained by entrapment of photosensitizer Photolon in polysiloxanes that was prepared by the sol-gel method. The material was characterized by its porosity, optical properties (fluorescence and absorbance), and laser-induced antimicrobial activity against Staphylococcus epidermidis, Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli. The permanent encapsulation of Photolon in the silica coating and the antimicrobial efficiency was confirmed by confocal microscope and digital holotomography. The generation of free radicals from nanoporous surfaces was proved by scanning Kelvin probe microscopy. For the first time, it was confirmed that Kelvin probe microscopy can be a label-free, noncontact alternative to other conventional methods based on fluorescence or chemiluminescence probes, etc. It was confirmed that the proposed photoactive coating enables the antibacterial photodynamic effect based on free radicals released from the surface of the coating. The highest bactericidal efficiency of the proposed coating was 87.16%. This coating can selectively limit the multiplication of bacterial cells, while protecting the environment and reducing the risk of surface contamination.

Three-Dimensional Augmented Reality Visualization Informs Locoregional Therapy in a Translational Model of Hepatocellular Carcinoma.

PURPOSE: To evaluate the utility of visualizing preprocedural MR images in 3-dimensional (3D) space using augmented reality (AR) before transarterial embolization of hepatocellular carcinoma (HCC) in a preclinical model. MATERIALS AND METHODS: A total of 28 rats with diethylnitrosamine-induced HCCs > 5 mm treated with embolization were included in a prospective study. In 12 rats, 3D AR visualization of preprocedural MR images was performed before embolization. Procedural metrics including catheterization time and radiation exposure were compared vs a prospective cohort of 16 rats in which embolization was performed without AR. An additional cohort of 15 retrospective cases was identified and combined with the prospective control cohort (n = 31) to improve statistical power. RESULTS: A 37% reduction in fluoroscopy time, from 11.7 min to 7.4 minutes, was observed with AR when compared prospectively, which did not reach statistical significance (P = .12); however, when compared with combined prospective and retrospective controls, the reduction in fluoroscopy time from 14.1 min to 7.4 minutes (48%) was significant (P = .01). A 27% reduction in total catheterization time, from 42.7 minutes to 31.0 minutes, was also observed with AR when compared prospectively, which did not reach statistical significance (P = .11). No significant differences were seen in dose-area product or air kerma prospectively. CONCLUSIONS: Three-dimensional AR visualization of preprocedural imaging may aid in the reduction of procedural metrics in a preclinical model of transarterial embolization. These data support the need for further studies to evaluate the potential of AR in endovascular oncologic interventions.

Physico-Chemical and Light-Induced Properties of Quinoline Azo-dyes Polymers.

We present investigation of optical and photochromic properties as well as of surface quality of thin films of novel methacrylic polymers with 8-hydroxyquinoline azo-dyes in side-chain. Additionally, thermal stability of polymer powders was examined and their glass transition temperature was determined. Optical properties (extinction coefficient and refractive index) were determined by spectroscopic ellipsometry (SE) combined with absorbance measurements. Photoresponsive behavior was investigated by determination of photoisomerization rates under irradiation with unpolarized 365 nm light, as well as by conduction of holographic grating inscription experiment. Thin film quality was determined by atomic force microscopy (AFM) measurements. Thermal analysis was performed by thermogravimetric (TG), derivative thermogravimetric (DTG) and differential scanning calorimetry (DSC) measurements. We found that optical properties as well as photoisomerization rates of investigated polymers are dependent on the substituent in the para position of the phenyl ring. Surface relief grating inscription was successfully generated only for materials with chromophores containing dimethylamino (N(CH3)2) and methyl (CH3) substituents, but all materials exhibited birefringence grating in the bulk. Surface of most thin films was very smooth, but its quality was impaired by neutral (H) as well as carboxyl (COOH) substituent. Thermal stability of copolymers with side-chain chromophores was improved compared to pure poly(methyl methacrylate) (PMMA).

Evidence of Skin Barrier Damage by Cyclic Siloxanes (Silicones)-Using Digital Holographic Microscopy.

Cyclic siloxanes (D4, D5, D6) are widely used in skin products. They improve skin sensory properties and alleviate dry skin, but there is still one report (published 2019), which regards their effects on the destruction of the skin barrier, by using fluorescence microscopy and attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR). A new skin-imaging technique, digital holographic microscopy (DHM), was used for the first time to investigate the impact of D4, D5, and D6 on the skin barrier. We observed irreversible damage of the stratum corneum due to the interaction with cyclic siloxanes. These substances changed: (a) the first level of the skin barrier through destabilization of the intercellular lipid lamellae and destruction of the corneocyte structure (measured with axial nanometer resolution), (b) the second level by collapse of not only corneocytes but also of a significant part of the clusters, leading to the loss of the stratum corneum integrity and formation of the lacunae, (c) the third level as an effect of the change in the surface geometrical topography of the stratum corneum and disruption of the integrity of this skin layer, measured with lateral micrometer resolution. DHM allowed also to identify an important pathway for substances to penetrate into the skin through canyons surrounding the clusters. Our investigations provide advanced information for understanding the mechanisms by which various substances pass the skin barrier, including uncontrolled diffusion into the skin.

Three-Dimensional Bacterial Motions near a Surface Investigated by Digital Holographic Microscopy: Effect of Surface Stiffness.

Surface stiffness plays a critical role in bacterial adhesion, but the mechanism is unclear since the bacterial motion before adhesion is overlooked. Herein, the three-dimensional (3D) motions of Escherichia coli and Pseudonomas sp. nov 776 onto poly(dimethylsiloxane) (PDMS) surfaces with varying stiffness before adhering were monitored by digital holographic microscopy (DHM). As Young's modulus (E) of the PDMS surface decreases from 278.1 to 3.4 MPa, the adhered E. coli and Pseudonomas sp. decrease in number by 40.4 and 34.9%, respectively. Atomic force microscopy (AFM) measurements show that the adhesion force of bacteria to the surface declines with the decreased surface stiffness. In contrast, a nontumbling mutant of adhered E. coli (HCB1414 with the adaptive function being partially deficient) decreases much less (by 18.4%). On the other hand, the tumble frequency (Ft) of E. coli HCB1 and flick frequency (Ff) of Pseudomonas sp. increase as the surface stiffness decreases, and the motion bias (Bθ) of Pseudomonas sp. also increases. These facts clearly indicate that the bacteria have adapted responses to the surface stiffness. RNA sequencing (RNA-seq) reveals that the downregulated Cph2 and CsrA as well as the upregulated GcvA of swimming E. coli HCB1 in bulk near the softer surface promote the bacterial motility.

Learning-based automatic sensing and size classification of microparticles using smartphone holographic microscopy.

The accurate and fast size classification of microparticles is important in environmental monitoring and biomedical applications. Conventional methods for sensing and classifying microparticles require bulky optical setups and generally show medium performance. Accordingly, the development of a portable and smart platform for accurate particle size classification is essential. In this study, we propose a new sensing platform for automatic identification of microparticle types through the synergistic integration of smartphone-based digital in-line holographic microscopy (DIHM) and machine-learning algorithms. The smartphone-based DIHM system consists of a coherent laser beam, a pinhole, a sample holder, a three-dimensional printed attachment, and a modified built-in smartphone camera module. The portable device has a physical dimension of 4 × 8 × 10 cm3 and 220 g in weight. Holograms of various polystyrene microparticles with different sizes (d = 2-50 µm) were recorded with a wide field-of-view and high spatial resolution. To establish a proper classification model, tens of features including geometrical parameters and light-intensity distributions were extracted from holograms of individual particles, and five machine-learning algorithms were used. After examining the performance of several classifiers, the resulting support vector machine model trained by using three geometrical parameters and three extracted parameters from light-intensity distributions shows the highest accuracy in the particle classification of the training and test sets (>98%). Therefore, the developed handheld smartphone-based platform can be potentially utilized to cope with various imaging needs in mobile healthcare and environmental monitoring.

Spatial Frequency Response of Epoxy-Based Volume Holographic Recording Material.

Holographic volume phase gratings are recorded in an epoxy-based, free-surface, volume holographic recording material. Light-induced gratings are formed by photo-triggered mass migration caused by component diffusion. The material resolution enables a wide range of pattern spacings, to record both transmission and reflection holograms with many different spatial frequencies. An optimum spatial frequency response is found between the low spatial frequency roll-off and the high spatial frequency cut-off. The influence of the energy density of exposure on the spatial frequency response is investigated. Secondary volume holographic gratings (parasitic gratings) are observed in the high frequency range. The possibility of distinguishing the regular grating from the secondary grating is discussed in the form of probe wavelength detuning.

Differential interference contrast microscopy for cells using hard x-ray holography.

We propose a differential interference contrast method for cells using hard x-ray Gabor holography and knife-edge filtering in the spatial frequency domain, without relying on beam shearing. A phase object is holographically recorded and reconstructed by computer. Interference between the wavefronts of zeroth order weighted by ejπ/2 in the positive frequency region produces a dark image. Similarly, interference between the wavefronts of the zeroth order weighted by ej3π/2 in the negative frequency region produces a bright image. By adding these two intensity distributions, good quality phase-contrast images of 8-µm-diameter polystyrene beads and human HeLa cells were obtained.

Applications of label-free, quantitative phase holographic imaging cytometry to the development of multi-specific nanoscale pharmaceutical formulations.

A label-free, high content, time-lapse holographic imaging system was applied to studies in pharmaceutical compound development. Multiple fields of cellular images are obtained over typically several day evaluations within standard CO2 incubators. Events are segmented to obtain population data of cellular features, which are displayed in scattergrams and histograms. Cell tracking is accomplished, accompanied by Cartesian plots of cell movement, as well as plots of cell features vs. time in novel 4-D displays of X position, Y position, time, and cell thickness. Our review of the instrument validation data includes 1) tracking of Giant HeLa cells, which may be undergoing neosis, a process of tumor stem cell generation; 2) tracking the effects of cell cycle related toxic agents on cell lines; 3) using MicroRNAs to reverse the polarization state in macrophages to induce tumor cell killing; 4) development of liposomal nanoformulations to overcome Multi-Drug Resistance (MDR) in ovarian cancer cells; and 5) development of dual sensitive micelles to specifically target matrix metalloproteinase 2 (MMP2) over-expressing cell lines. © 2017 International Society for Advancement of Cytometry.

Plasmonic nanoparticle scattering for color holograms.

This work presents an original approach to create holograms based on the optical scattering of plasmonic nanoparticles. By analogy to the diffraction produced by the scattering of atoms in X-ray crystallography, we show that plasmonic nanoparticles can produce a wave-front reconstruction when they are sampled on a diffractive plane. By applying this method, all of the scattering characteristics of the nanoparticles are transferred to the reconstructed field. Hence, we demonstrate that a narrow-band reconstruction can be achieved for direct white light illumination on an array of plasmonic nanoparticles. Furthermore, multicolor capabilities are shown with minimal cross-talk by multiplexing different plasmonic nanoparticles at subwavelength distances. The holograms were fabricated from a single subwavelength thin film of silver and demonstrate that the total amount of binary information stored in the plane can exceed the limits of diffraction and that this wavelength modulation can be detected optically in the far field.

Digital holographic microscopy as a tool to study the thermal shape fluctuations of lipid vesicles.

The bending elasticity modulus of lipid membranes is obtained by applying for the first time, to the best of our knowledge, a novel experimental technique based on digital holographic microscopy. The fluctuations of the radius with time were extracted by tracking and measuring the optical thickness at the vesicle poles. The temporal autocorrelation function of the vesicle diameter computed for each of the studied vesicles was then fitted with the theoretical expression to deduce the membrane's tension and bending constant. For the bending elasticity modulus of SOPC bilayers, the value of (0.93 ± 0.03) × 10(-12) erg was obtained. This result is in accordance with values previously obtained by means of other conventional methods for the same type of lipid membrane in the presence of sugar molecules in aqueous medium. The obtained results encourage the future development of the digital holographic microscopy as a technique suitable for the measurement of the bending elasticity of lipid membranes.

Optofluidic bioimaging platform for quantitative phase imaging of lab on a chip devices using digital holographic microscopy.

We propose a versatile 3D phase-imaging microscope platform for real-time imaging of optomicrofluidic devices based on the principle of digital holographic microscopy (DHM). Lab-on-chip microfluidic devices fabricated on transparent polydimethylsiloxane (PDMS) and glass substrates have attained wide popularity in biological sensing applications. However, monitoring, visualization, and characterization of microfluidic devices, microfluidic flows, and the biochemical kinetics happening in these devices is difficult due to the lack of proper techniques for real-time imaging and analysis. The traditional bright-field microscopic techniques fail in imaging applications, as the microfluidic channels and the fluids carrying biological samples are transparent and not visible in bright light. Phase-based microscopy techniques that can image the phase of the microfluidic channel and changes in refractive indices due to the fluids and biological samples present in the channel are ideal for imaging the fluid flow dynamics in a microfluidic channel at high resolutions. This paper demonstrates three-dimensional imaging of a microfluidic device with nanometric depth precisions and high SNR. We demonstrate imaging of microelectrodes of nanometric thickness patterned on glass substrate and the microfluidic channel. Three-dimensional imaging of a transparent PDMS optomicrofluidic channel, fluid flow, and live yeast cell flow in this channel has been demonstrated using DHM. We also quantify the average velocity of fluid flow through the channel. In comparison to any conventional bright-field microscope, the 3D depth information in the images illustrated in this work carry much information about the biological system under observation. The results demonstrated in this paper prove the high potential of DHM in imaging optofluidic devices; detection of pathogens, cells, and bioanalytes on lab-on-chip devices; and in studying microfluidic dynamics in real time based on phase changes.

In-line digital holographic imaging in volume holographic microscopy.

A dual-plane in-line digital holographic imaging method incorporating volume holographic microscopy (VHM) is presented to reconstruct objects in a single shot while eliminating zero-order and twin-image diffracted waves. The proposed imaging method is configured such that information from different axial planes is acquired simultaneously using multiplexed volume holographic imaging gratings, as used in VHM, and recorded as in-line holograms where the corresponding reference beams are generated in the fashion of Gabor's in-line holography. Unlike conventional VHM, which can take axial intensity information only at focal depths, the proposed method digitally reconstructs objects at any axial position. Further, we demonstrate the proposed imaging technique's ability to effectively eliminate zero-order and twin images for single-shot three-dimensional object reconstruction.

Cell shape identification using digital holographic microscopy.

We present a cost-effective, simple, and fast digital holographic microscopy method based upon Rayleigh-Sommerfeld backpropagation for identification of the geometrical shape of a cell. The method was tested using synthetic hologram images generated by ray-tracing software and from experimental images of semitransparent spherical beads and living red blood cells. Our results show that, by only using the real part of the back-reconstructed amplitude, the proposed method can provide information of the geometrical shape of the object and at the same time accurately determine the axial position of the object under study. The proposed method can be used in flow chamber assays for pathophysiological studies where fast morphological changes of cells are studied in high numbers and at different heights.

Investigation of (Fe,Co)NbB-Based Nanocrystalline Soft Magnetic Alloys by Lorentz Microscopy and Off-Axis Electron Holography.

The relationship between microstructure and magnetic properties of a (Fe,Co)NbB-based nanocrystalline soft magnetic alloy was investigated by analytical transmission electron microscopy (TEM). The microstructures of (Fe0.5Co0.5)80Nb4B13Ge2Cu1 nanocrystalline alloys annealed at different temperatures were characterized by TEM and electron diffraction. The magnetic structures were analyzed by Lorentz microscopy and off-axis electron holography, including quantitative measurement of domain wall width, induction, and in situ magnetic domain imaging. The results indicate that the magnetic domain structure and particularly the dynamical magnetization behavior of the alloys strongly depend on the microstructure of the nanocrystalline alloys. Smaller grain size and random orientation of the fine particles decrease the magneto-crystalline anisotropy and suggests better soft magnetic properties which may be explained by the anisotropy model of Herzer.

Non-iterative holographic axial localization using complex amplitude of diffraction-free vortices.

We present a novel technique of digital holography using digitally implemented diffraction-free vortices for a precise three-dimensional (3D) localization of point-like objects. The localization is realized by the processing of the holographic image reconstructed at arbitrarily selected plane. Separating a single radial component of the spatial spectrum and modulating its phase by a virtual spiral mask, the holographic images of individual object points are transformed to the image structures analogous to the diffraction-free vortex beams. The real part of the complex amplitude of the digital vortices creates the shape-invariant patterns rotating due to a defocusing. Determining the angular rotation, the axial positions of the individual point objects are specified over a wide axial range. In the proposed method, a single in-line hologram is processed without phase shifting and multiplane reconstruction, so that a dynamic localization and tracking of particles becomes possible. The principle of the method is presented in a unified computational model valid for both coherent and incoherent techniques of digital holography. The functionality of the method has been verified in experiments of the Fresnel incoherent correlation holography (FINCH) and its flexibility presented by controlled variations of the localization sensitivity. The application potential has been demonstrated by the defocusing image rotation of fixed fluorescent microspheres and the 3D localization and tracking of moving polystyrene beads resulting in the trajectory reconstruction of a selected particle.

Biomedical applications of holographic microspectroscopy [invited].

The identification and quantification of specific molecules are crucial for studying the pathophysiology of cells, tissues, and organs as well as diagnosis and treatment of diseases. Recent advances in holographic microspectroscopy, based on quantitative phase imaging or optical coherence tomography techniques, show promise for label-free noninvasive optical detection and quantification of specific molecules in living cells and tissues (e.g., hemoglobin protein). To provide important insight into the potential employment of holographic spectroscopy techniques in biological research and for related practical applications, we review the principles of holographic microspectroscopy techniques and highlight recent studies.