Microscopy Imaging Methods Research Articles

Quantitative analysis of DNA-Dox diffusion kinetics in a microfluidic device using the fluorescence lifetime imaging microscopy method

We used a fluorescence lifetime imaging microscopy combined with a microfluidic mixer to study the kinetics of doxorubicin hydrochloride (Dox) diffusion into DNA. The mixing process in the microchannel model was simulated using COMSOL. According to the change of fluorescence lifetime before and after the binding of Dox to DNA, the fluorescence quenching effect of DNA on Dox was obtained both temporally and spatially, and the kinetic information was analyzed from imaging experiments. The experimental results show good agreement with the computational fluid dynamics simulation, and this method could be used to analyze the diffusion process of drug molecules.

Pressure drop analysis for the droplet break-up flow in a locally constrictive microchannel

Locally constrictive microchannels are effective components to implement droplet break-up in microfluidic processes. We herein report an experimental study of droplet break-up flow based on pressure drop analysis. The time varying pressure drops were recorded by a self-assembled experimental platform with measurement accuracy of 1 Pa, and the variations of those pressure drops were in accord with the fluctuations of capillary pressure. Fourier transform was then employed to analyze the droplet break-up behaviors and confirm the numbers of daughter droplets instead of traditional microscopic imaging method. With the guidance of the pressure drop analysis, the effects of crucial factors such as surfactant concentration, fluid flow rate, and fluid viscosity on droplet break-up were investigated. The additional hydrostatic energy cost for droplet stretching and deformation in the constriction was finally discussed in contrast to the increase of interfacial energy, which were both confirmed on the level of 10−8 J/droplet.

Assembly of Building Blocks by Double-End-Anchored Polymers in the Dilute Regime Mediated by Hydrophobic Interactions at Controlled Distances.

Hierarchical assembly of building blocks via competing, orthogonal interactions is a hallmark of many of nature's composite materials that do not require highly specific ligand-receptor interactions. To mimic this assembly mechanism requires the development of building blocks capable of tunable interactions. In the present work, we explored the interplay between repulsive (steric and electrostatic) and attractive hydrophobic forces. The designed building blocks allow hydrophobic forces to effectively act at controlled, large distances, to create and tune the assembly of membrane-based building blocks under dilute conditions, and to affect their interactions with cellular membranes via physical cross-bridges. Specifically, we employed double-end-anchored poly(ethylene glycol)s (DEA-PEGs)-hydrophilic PEG tethers with hydrophobic tails on both ends. Using differential-interference-contrast optical microscopy, synchrotron small-angle X-ray scattering (SAXS), and cryogenic electron microscopy, we investigated the ability of DEA-PEGs to mediate assembly in the dilute regime on multiple length scales and on practical time scales. The PEG length, anchor hydrophobicity, and molar fraction of DEA-PEG molecules within a membrane strongly affect the assembly properties. Additional tuning of the intermembrane interactions can be achieved by adding repulsive interactions via PEG-lipids (steric) or cationic lipids to the DEA-PEG-mediated attractions. While the optical and electron microscopy imaging methods provided qualitative evidence of the ability of DEA-PEGs to assemble liposomes, the SAXS measurements and quantitative line-shape analysis in dilute preparations demonstrated that the ensemble average of loosely organized liposomal assemblies maintains DEA-PEG concentration-dependent tethering on defined nanometer length scales. For cationic liposome-DNA nanoparticles (CL-DNA NPs), aggregation induced by DEA-PEGs decreased internalization of NPs by cells, but tuning the DEA-PEG-induced attractions by adding repulsive steric interactions via PEG-lipids limited aggregation and increased NP uptake. Furthermore, confocal microscopy imaging together with colocalization studies with Rab11 and LysoTracker as markers of intracellular pathways showed that modifying CL-DNA NPs with DEA-PEGs alters their interactions with the plasma and endosomal membranes.

A correlation analysis of Light Microscopy and X-ray MicroCT imaging methods applied to archaeological plant remains\u2019 morphological attributes visualization

In this work, several attributes of the internal morphology of drupaceous fruits found in the archaeological site Monte Castelo (Rondonia, Brazil) are analyzed by means of two different imaging methods. The aim is to explore similarities and differences in the visualization and analytical properties of the images obtained via High Resolution Light Microscopy and X-ray micro-computed tomography (X-ray MicroCT) methods. Both provide data about the three-layered pericarp (exo-, meso- and endocarp) of the studied exemplars, defined by cell differentiation, vascularisation, cellular contents, presence of sclerenchyma cells and secretory cavities. However, it is possible to identify a series of differences between the information that can be obtained through each of the methods. These variations are related to the definition of contours and fine details of some characteristics, their spatial distribution, size attributes, optical properties and material preservation. The results obtained from both imaging methods are complementary, contributing to a more exhaustive morphological study of the plant remains. X-ray MicroCT in phase-contrast mode represents a suitable non-destructive analytic technique when sample preservation is required.

Nucleation, growth, and superlattice formation of nanocrystals observed in liquid cell transmission electron microscopy

Abstract

Dynamics Characterization of the Acoustically Driven Single Microbubble near the Rigid and Elastic Wall

The dynamic behaviors of single acoustic bubble are the key fundamental problem in exploring the mechanism of acoustic cavitation. In this paper, a synchronous high-speed microscopic imaging method is proposed to clearly record the temporary evolution of single bubble in low-frequency ultrasonic field. In the experiment, the temporal evolution of the bubble with two different initial radii in an ultrasonic field are recorded by the high-speed camera at 300 000 frames per second, and other important characteristics of the bubbles are calculated and analyzed. In these experiments, the single bubbles behave similar under the same relative distance from a rigid wall, but the dynamic behaviors of the bubble with different initial radii have obvious difference. In addition, the bubble dynamics of the bubble near an elastic wall are also investigated and compared to the bubble near the rigid wall. It is found that the elasticity would significantly influence the dynamic characteristics during bubble collapse and rebound. In this work, the synchronous high-speed microscopic imaging method demonstrates the abilities to experimentally investigate the rapidly dynamics of single bubble in ultrasonic field.

Snapshot multifocal light field microscopy.

Light field microscopy (LFM) is an emerging technology for high-speed wide-field 3D imaging by capturing 4D light field of 3D volumes. However, its 3D imaging capability comes at a cost of lateral resolution. In addition, the lateral resolution is not uniform across depth in the light field dconvolution reconstructions. To address these problems, here, we propose a snapshot multifocal light field microscopy (MFLFM) imaging method. The underlying concept of the MFLFM is to collect multiple focal shifted light fields simultaneously. We show that by focal stacking those focal shifted light fields, the depth-of-field (DOF) of the LFM can be further improved but without sacrificing the lateral resolution. Also, if all differently focused light fields are utilized together in the deconvolution, the MFLFM could achieve a high and uniform lateral resolution within a larger DOF. We present a house-built MFLFM system by placing a diffractive optical element at the Fourier plane of a conventional LFM. The optical performance of the MFLFM are analyzed and given. Both simulations and proof-of-principle experimental results are provided to demonstrate the effectiveness and benefits of the MFLFM. We believe that the proposed snapshot MFLFM has potential to enable high-speed and high resolution 3D imaging applications.

Crossed-beam pump-probe microscopy

We present a new imaging method for pump-probe microscopy that explores non-collinear excitation. This method (crossed-beam pump-probe microscopy, or CBPM) can significantly improve the axial resolution when imaging through low-NA lenses, providing an alternative way for depth-resolved, large field-of-view imaging. We performed a proof-of-concept demonstration, characterized CBPM's resolution using different imaging lenses, and measured an enhanced axial resolution for certain types of low-NA lenses.

Hyperspectral Microscopic Imaging for Automatic Detection of Head and Neck Squamous Cell Carcinoma Using Histologic Image and Machine Learning.

The purpose of this study is to develop hyperspectral imaging (HSI) for automatic detection of head and neck cancer cells on histologic slides. A compact hyperspectral microscopic system is developed in this study. Histologic slides from 15 patients with squamous cell carcinoma (SCC) of the larynx and hypopharynx are imaged with the system. The proposed nuclei segmentation method based on principle component analysis (PCA) can extract most nuclei in the hyperspectral image without extracting other sub-cellular components. Both spectra-based support vector machine (SVM) and patch-based convolutional neural network (CNN) are used for nuclei classification. CNNs were trained with both hyperspectral images and pseudo RGB images of extracted nuclei, in order to evaluate the usefulness of extra information provided by hyperspectral imaging. The average accuracy of spectra-based SVM classification is 68%. The average AUC and average accuracy of the HSI patch-based CNN classification is 0.94 and 82.4%, respectively. The hyperspectral microscopic imaging and classification methods provide an automatic tool to aid pathologists in detecting SCC on histologic slides.

High-resolution quantification of discrete phagocytic events by live cell time-lapse high-content microscopy imaging.

Phagocytosis is a dynamic process central to immunity and tissue homeostasis. Current methods for quantification of phagocytosis largely rely on indirect or static measurements, such as target clearance or dye uptake, and thus provide limited information about engulfment rates or target processing. Improved kinetic measurements of phagocytosis could provide useful, basic insights in many areas. We present a live-cell, time-lapse and high-content microscopy imaging method based on the detection and quantification of fluorescent dye 'voids' within phagocytes that result from target internalization to quantify phagocytic events with high temporal resolution. Using this method, we measure target cell densities and antibody concentrations needed for optimal antibody-dependent cellular phagocytosis. We compare void formation and dye uptake methods for phagocytosis detection, and examine the connection between target cell engulfment and phagolysosomal processing. We demonstrate how this approach can be used to measure distinct forms of phagocytosis, and changes in macrophage morphology during phagocytosis related to both engulfment and target degradation. Our results provide a high-resolution method for quantifying phagocytosis that provides opportunities to better understand the cellular and molecular regulation of this fundamental biological process.

Terahertz Imaging Patch Antenna for Cancer Diagnosis Applications

Cancer is the world second most common syndrome which causes death. In statistics nearly one of every four death occurs day-to-day, but it is remediable if detected earlier. The numerous imaging technologies exist for diagnosis with different constrains but those techniques provide either macroscopic or microscopic imaging. Histopathology of biopsy samples uses microscopic imaging methods to provide corporeal and functional information. In macroscopic level X-ray and MRI are used to provide images of living tissues and can achieve only much poorer resolution and specificity. Moreover it is harmful in terms of radiation and other causes To syndicate the macroscopic and microscopic imaging progresses, for delineating the precise margins of cancers is one of the prime mysterious complication in medical imaging.The THztomography research does that syndication and achieves much high resolution with Non-ionizing radiation. The proposed nanomaterial based Microstrip antenna is pertinent to do THztomography with low cost and high resolution which can diagnosis and detection various cancers such as skin cancer, breast cancer, cervical cancer and colon cancer. In this paper, a terahertz imaging antenna was designed and analyzed using Ansys - HFSS v.14 simulation tool.

The retreatment abilities of ProTaper Next and F6 Skytaper: a micro-computed tomography study.

The aim of this study was to evaluate the retreatment abilities of the ProTaper Next (PTN) and F6 SkyTaper (F6) systems by using micro-computed tomography (microct), radiographic and microscopic imaging techniques. The root canals of twenty-six extracted mandibular premolar teeth were prepared and obturated. For the retreatment procedure, the teeth were randomly divided into two equal groups according to endodontic instruments: PTN (X4) and F6 (#40/.06). Pre- and post-operative filling material volumes were measured with micro-ct, and areas were measured with radiographic and microscopic imaging techniques. The percentages of residual material were calculated, and then statistically compared. The significance level was set at p<0.05. There was no statistically significant difference between F6 and PTN for retreatment efficacy in the micro-ct and radiographic imaging techniques (p>0.05). PTN demonstrated better cleaning ability when evaluated by microscopic imaging. (p<0.05). The correlation was moderate between micro-ct and radiographic, and micro-ct and microscopic imaging groups; however, it was strong between radiographic and microscopic imaging methods. The PTN and F6 files had similar effects in the removal of filling material with microct evaluation. The radiographic imaging method gave similar results with micro-ct imaging.

Prediction of liquid jet trajectory in supersonic crossflow and continuous liquid column model

The trajectory of the spray is studied theoretically and experimentally when a round liquid jet is injected into a supersonic crossflow vertically. A solid model of continuous liquid column is established in three-dimensional space. The cross-section deformation equation of the continuous liquid column along the injection direction is established using a method of micro-element analysis. The stress analysis of cross section is simplified into a two-dimensional droplet. The shape of the cross section is considered to continuously change from circular to elliptical shape. And the bow shock wave in front of the jet column is simplified into an oblique shock wave with a known shock angle. Based on this, the calculation of aerodynamic force is greatly simplified. A dimensionless parameter named effective deformation time of liquid column (the logogram is &lt;inline-formula&gt;&lt;tex-math id="M300"&gt;\begin{document}${t_{\rm valid}}$\end{document}&lt;/tex-math&gt;&lt;alternatives&gt;&lt;graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="23-20200903_M300.jpg"/&gt;&lt;graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="23-20200903_M300.png"/&gt;&lt;/alternatives&gt;&lt;/inline-formula&gt;) is defined and used to judge the end point of the liquid column quantitatively. The liquid jet trajectory and cross-section deformation can be calculated using MATLAB software. The instantaneous images of continuous liquid columns in supersonic crossflow are captured using high-spatial-resolution microscopic imaging methods. The microscopic imaging system is composed of a double pulse solid-state laser, computer, CCD camera, synchronous controller, microscope lens and laser diffuser. After passing through the laser diffuser, a plane background light with uniform distribution is formed on the scattering plate. The mean filtering method is used to filter the original image. After filtering, the range of gray distribution in the background area is obviously reduced. The distribution of gray value is more concentrated, and the background of the image is more uniform. Then the image edge detection function is used to obtain the near-field jet trajectory. The parameter variables studied include liquid injection pressure drop (1–2 MPa), liquid nozzle diameter (0.5 mm/1.0 mm), and liquid gas momentum ratio (3.32–7.27). The results show that the continuous liquid column model can better predict the jet trajectory on the center plane and the shape of the liquid column in three-dimensional space. It is indicated that the predictive result matches well with the experimental result. This study is of great significance for establishing the solid-particle coupling model of liquid jet in supersonic crossflows.

IO&lt;sub&gt;2&lt;/sub&gt;-Supported Single-Atom Catalysts for Photocatalytic Reactions

Titania（TiO2） has been among the most widely investigated and used metal oxides over the past years, as it has various functional applications. Extensive research into TiO2 and industrial interest in this material have been triggered by its high abundance, excellent corrosion resistance, and low cost. To improve the activity of TiO2 in heterogeneous catalytic reactions, noble metals are used to accelerate the reactions. However, in the case of nanoparticles supported on TiO2, the active sites are usually limited to the peripheral sites of the noble metal particles or at the interface between the particle and the support. Thus, highly dispersed single metal atoms are desired for the effective utilization of precious noble metals. The study of oxide-supported isolated atoms, the so-called single-atom catalysts（SACs）, was pioneered by Zhang’s group. The high dispersion of precious noble metals results helps reduce the cost associated with catalyst preparation. Because of the presence of active centers as single atoms, the deactivation of metal atoms during the reaction, e.g., by coking for large agglomerates, is retarded. The unique coordination environment of the noble metal center provides special sites for the reaction, consequently increasing the selectivity of the reaction, including the enantioselectivity and stereoselectivity. Hence, supported SACs can bridge homogenous and heterogeneous reactions in solution as they provide selective reaction sites and are recyclable. Moreover, owing to the high site homogeneity of the isolated metal atoms, SACs are ideal models for establishing the structure-activity relationships. The present review provides an overview of recent works on the synthesis, characterization, and photocatalytic applications of SACs（Pt1, Pd1, Ir1, Rh1, Cu1, Ru1） supported on TiO2. The preparation of single atoms on TiO2 includes the creation of surface defective sites, surface modification, stabilization by high-temperature shockwave treatment, and metal-ligand self-assembly. Conventional characterization methods are categorized as microscopic imaging and spectroscopic methods, such as aberration-corrected scanning transmission electron microscopy（STEM）, scanning tunneling microscopy（STM）, extended X-ray absorption fine structure analysis（EXAFS）, and diffuse reflectance infrared Fourier transform spectroscopy（DRIFTS）. We attempted to address the critical factors that lead to the stabilization of single-metal atoms on TiO2, and elucidate the mechanism underlying the photocatalytic hydrogen evolution and CO2 reduction. Although many fascinating applications of TiO2-supported SACs in photocatalysis could only be addressed superficially and in a referencing manner, we hope to provide interested readers with guidelines based on the wide literature, and more specifically, to provide a comprehensive overview of TiO2-supported SACs.

Fast structured illumination three-dimensional color microscopic imaging method based on Hilbert-transform

As a wide-field microscopy, structured illumination microscopy (SIM) enables super-resolution and three-dimensional (3D) imaging. It has recently received lots of attention due to the advantages of high spatial resolution, short image recording time, and less photobleaching and phototoxicity. The SIM has found numerous important applications in time-lapse imaging of living tissues and cellular structures in the field of biomedical science. Color information is an important physical quantity describing the characteristics of living creatures and reflects the differences in its microstructure and optical property to some extent. Although HSV (hue, saturation, value) color space based structured illumination full-color 3D optical sectioning technique can recover the full color information on the surface of the samples without color distortion. However, for each optical sectioning, three raw images with fixed phase shift are required to calculate the sectioning images by the rootmean square (RMS) algorithm. This will dramatically increase the data acquisition time and data storage space, especially for a large-scaled sample that needs image stitching strategy. The image processing progress operated in HSV color space need to run the RMS algorithm three times in each channel of HSV space for every section, and transform the images between RGB (red-green-blue) space and HSV space twice. This will absolutely extend the data processing time and put forward higher requirements for computer hardware and software for data storage and processing. To this end, in this paper, a fast 3D color optical sectioning SIM algorithm based on Hilbert-transform is proposed. The Hilbert-transform has proved to be a powerful tool in digital signal and image processing and has successfully applied to the SIM. Here, only two raw images with structured illumination are needed to reconstruct a full-color optical sectioned image for each slice. This fast 3D color sectioning method has the advantage of insensitivity to phase-shift error and has better adaptability to noise, high quality color sectioning images can be obtained under the phase-shift error or noise disturbed environment. The image acquisition data are reduced by 1/3 and the color optical sectioning reconstruction time is saved by about 28%, this new method effectively improves the efficiency and speed for 3D color imaging and will bring a wider application range for SIM.

The impact of age, mineralization, and collagen orientation on the mechanics of individual osteons from human femurs

Osteons are the end product of cortical bone remodeling. Changes in their mechanical properties may be an indicator of overall bone health. Aszenci and Bonucci first evaluated the compressive properties of individual osteons in 1968. However, these results have never been independently validated and technological advances allow for further investigation of osteon mechanics. In the present study, an experimental protocol was established such that osteons ~250 µm in diameter were successfully extracted from human cortical bone (males, ages 93 and 64) and loaded in compression. Both micro-computed tomography and second harmonic generation (SHG) microscopy imaging methods were incorporated into the protocol to improve the structural and compositional assessment of each osteon. Further, a high-resolution videography system was used to monitor the osteons during mechanical testing, permitting insight into deformation mechanisms in situ. The results show that the older donor osteons (male, age 93 (n = 13)) were stiffer (p = 0.04) and more highly mineralized (p = 0.03), while the younger donor osteons (male, 64 (n = 14)) exhibited more heterogeneity in the measured mechanical properties with the presence of both stiff and more compliant osteons. SHG intensity indicated predominantly longitudinal fiber alignment for all osteons while the videography data revealed three distinct failure modes and confirmed that whitening observed during yielding of macroscale bone specimens represents failure at the local level. The stiffness and strength of the younger donor osteons were heavily dependent on the gross structure of the osteon, while these properties were dominated by collagen orientation in the older donor osteons.

Digital refocusing and extended depth of field reconstruction in Fourier ptychographic microscopy.

Fourier ptychography microscopy (FPM) is a recently developed microscopic imaging method that allows the recovery of a high-resolution complex image by combining a sequence of bright and darkfield images acquired under inclined illumination. The capacity of FPM for high resolution imaging at low magnification makes it particularly attractive for applications in digital pathology which require imaging of large specimens such as tissue sections and blood films. To date most applications of FPM have been limited to imaging thin samples, simplifying both image reconstruction and analysis. In this work we show that, for samples of intermediate thickness (defined here as less than the depth of field of a raw captured image), numerical propagation of the reconstructed complex field allows effective digital refocusing of FPM images. The results are validated by comparison against images obtained with an equivalent high numerical aperture objective lens. We find that post reconstruction refocusing (PRR) yields images comparable in quality to adding a defocus term to the pupil function within the reconstruction algorithm, while reducing computing time by several orders of magnitude. We apply PRR to visualize FPM images of Giemsa-stained peripheral blood films and present a novel image processing pipeline to construct an effective extended depth of field image which optimally displays the 3D sample structure in a 2D image. We also show how digital refocusing allows effective correction of the chromatic focus shifts inherent to the low magnification objective lenses used in FPM setups, improving the overall quality of color FPM images.

Axial high-resolution differential confocal microscopy

In this paper, an axial high-resolution differential confocal microscopy (AHDCM) is proposed to improve the axial resolution of confocal microscopy (CM). AHDCM divides the detection optical path of the confocal system into three paths: focal-plane detection imaging optical path, pre-focal detection imaging optical path and post-focal detection imaging optical path. The pre-focal and the post-focal detection imaging optical paths have the equal axial detector offset oriented in opposite direction. Subsequently, the axial resolution of the confocal system is improved by subtracting the sum of the pre-focal and post-focal detection signals from the twice focal-plane detection signal. Theoretical analyses and preliminary experiments indicate that, for an emitting wavelength of λ = 405 nm and numerical aperture of NA = 0.8, the AHDCM’s axial resolution improves by more than 35% compared with that of CM. Therefore, AHDCM is a practical and feasible new method for axial high-resolution microscopic imaging.

Subvisible Particulate Contamination in Cell Therapy Products-Can We Distinguish?

Cell therapy products represent an exciting new class of medicinal products, which must be parenterally administered. Thus, compliance with parenteral preparation guidelines is required. One requirement for parenteral products is the characterization of particle contaminations. As cell-based products are turbid suspensions, containing particles, the cells, characterization and control of foreign particle impurities remain a challenge. Within this study, we evaluated a flow imaging microscopy method for the detection and characterization of subvisible particle contaminations in cell-based products. We found that flow imaging microscopy is a potential method where subvisible particle contaminations can be differentiated from the cells in cell therapy products.

Electrode-free nanopore sensing by DiffusiOptoPhysiology.

A wide variety of single molecules can be identified by nanopore sensing. However, all reported nanopore sensing applications result from the same measurement configuration adapted from electrophysiology. Although urgently needed in commercial nanopore sequencing, parallel electrophysiology recording is limited in its cost and its throughput due to the introduced complexities from electronic integration. We present the first electrode-free nanopore sensing method defined as DiffusiOptoPhysiology (DOP), in which single-molecule events are monitored optically without any electrical connections. Single-molecule sensing of small molecules, macromolecules, and biomacromolecules was subsequently demonstrated. As a further extension, a fingertip-sized, multiplexed chip with single-molecule sensing capabilities has been introduced, which suggests a new concept of clinical diagnosis using disposable nanopore sensors. DOP, which is universally compatible with all types of channels and a variety of fluorescence imaging platforms, may benefit diverse areas such as nanopore sequencing, drug screening, and channel protein investigations.