RESUMEN

X-rays can penetrate deeply into biological cells and thus allow for examination of their internal structures with high spatial resolution. In this study, X-ray phase-contrast imaging and tomography is combined with an X-ray-compatible optical stretcher and microfluidic sample delivery. Using this setup, individual cells can be kept in suspension while they are examined with the X-ray beam at a synchrotron. From the recorded holograms, 2D phase shift images that are proportional to the projected local electron density of the investigated cell can be calculated. From the tomographic reconstruction of multiple such projections the 3D electron density can be obtained. The cells can thus be studied in a hydrated or even living state, thus avoiding artifacts from freezing, drying or embedding, and can in principle also be subjected to different sample environments or mechanical strains. This combination of techniques is applied to living as well as fixed and stained NIH3T3 mouse fibroblasts and the effect of the beam energy on the phase shifts is investigated. Furthermore, a 3D algebraic reconstruction scheme and a dedicated mathematical description is used to follow the motion of the trapped cells in the optical stretcher for multiple rotations.

RESUMEN

Dual-pulsed (DPL) laser deposition using oyster shells as targets was studied in order to find out if this method can replace the use of high-power pulsed lasers. Aspects related to changes in the morphological structure of the thin layer but also to the chemical composition of the obtained thin layer were analyzed and compared with the target as well as with the thin layers obtained with a higher power pulsed laser in a single-pulsed (SPL) regime. Orthorhombic structures were noticed with Scanning Electron Microscopy for the thin film obtained in DPL mode compared to the irregular particles obtained in SPL mode. The deacetylation process during ablation was evidenced by Fourier Transform Infrared spectroscopy, resulting in chitosan-based thin films. The effect of the obtained thin films of chitosan on the cells of baker's yeast (Saccharomyces cerevisiae) was studied. Restoration of the yeast paste into initial yeast was noticed mainly when the hemp fabric was used as support for the coating with yeas which was after that coated with chitosan thin film produced by DPL method.

RESUMEN

The relationship between the contrast to noise ratio and intensity based cross-correlation coefficients for both protein crystallography and X-ray imaging are compared. It is concluded that, for protein crystallography at near atomic resolution, the intensity based cross-correlation coefficients give a reasonable indication of the quality of the corresponding electron density. For X-ray imaging of biological materials such as cells and soft tissue, the wide range of contrast of the features means that intensity based correlation coefficients can give a poor indication of the interpretability of an image. Rather than the term resolution, it is the contrast to noise ratio for a feature of interest at the relevant spatial frequency that is more relevant. Additional metrics are required to describe the quality of an image, and these are discussed.

RESUMEN

BACKGROUND: The cultivation, analysis, and isolation of single cells or cell cultures are fundamental to modern biological and medical processes. The novel LIFTOSCOPE technology aims to integrate analysis and isolation into one versatile, fully automated device. METHODS: LIFTOSCOPE's three core technologies are high-speed microscopy for rapid full-surface imaging of cell culture vessels, AI-based semantic segmentation of microscope images for localization and evaluation of cells, and laser-induced forward transfer (LIFT) for contact-free isolation of cells and cell clusters. LIFT transfers cells from a standard microtiter plate (MTP) across an air gap to a receiver plate, from where they can be further cultivated. The LIFT laser is integrated into the optical path of an inverse microscope, allowing to switch quickly between microscopic observation and cell transfer. RESULTS: Tests of the individual process steps prove the feasibility of the concept. A prototype setup shows the compatibility of the microscope stage with the LIFT laser. A specifically designed MTP adapter to hold a receiver plate has been designed and successfully used for material transfers. A suitable AI algorithm has been found for cell selection. CONCLUSION: LIFTOSCOPE speeds up cell cultivation and analysis with a target process time of 10 minutes, which can be achieved if the cell transfer is sped up using a more efficient path-finding algorithm. Some challenges remain, like finding a suitable cell transfer medium. SIGNIFICANCE: The LIFTOSCOPE system can be used to extend existing cell cultivation systems and microscopes for fully automated biotechnological applications.

RESUMEN

Generation of amplified stimulated emission inside mammalian cells has paved the way for a novel bioimaging and cell sensing approach. Single cells carrying gain media (e.g., fluorescent molecules) are placed inside an optical cavity, allowing the production of intracellular laser emission upon sufficient optical pumping. Here, we investigate the possibility to trigger another amplified emission phenomenon (i.e., amplified spontaneous emission or ASE) inside two different cell types, namely macrophage and epithelial cells from different species and tissues, in the presence of a poorly reflecting cavity. Furthermore, the resulting ASE properties can be enhanced by introducing plasmonic nanoparticles. The presence of gold nanoparticles (AuNPs) in rhodamine 6G-labeled A549 epithelial cells results in higher intensity and lowered ASE threshold in comparison to cells without nanoparticles, due to the effect of plasmonic field enhancement. An increase in intracellular concentration of AuNPs in rhodamine 6G-labeled macrophages is, however, responsible for the twofold increase in the ASE threshold and a reduction in the ASE intensity, dominantly due to a suppressed in and out-coupling of light at high nanoparticle concentrations.

Asunto(s)

RESUMEN

The article elucidates the physical mechanism behind the generation of superior-contrast and high-resolution label-free images using an optical waveguide. Imaging is realized by employing a high index contrast multi-moded waveguide as a partially coherent light source. The modes provide near-field illumination of unlabeled samples, thereby repositioning the higher spatial frequencies of the sample into the far-field. These modes coherently scatter off the sample with different phases and are engineered to have random spatial distributions within the integration time of the camera. This mitigates the coherent speckle noise and enhances the contrast (2-10) × as opposed to other imaging techniques. Besides, the coherent scattering of the different modes gives rise to fluctuations in intensity. The technique demonstrated here is named chip-based Evanescent Light Scattering (cELS). The concepts introduced through this work are described mathematically and the high-contrast image generation process using a multi-moded waveguide as the light source is explained. The article then explores the feasibility of utilizing fluctuations in the captured images along with fluorescence-based techniques, like intensity-fluctuation algorithms, to mitigate poor-contrast and diffraction-limited resolution in the coherent imaging regime. Furthermore, a straight waveguide is demonstrated to have limited angular diversity between its multiple modes and therefore, for isotropic sample illumination, a multiple-arms waveguide geometry is used. The concepts introduced are validated experimentally via high-contrast label-free imaging of weakly scattering nanosized specimens such as extra-cellular vesicles (EVs), liposomes, nanobeads and biological cells such as fixed and live HeLa cells.

RESUMEN

The effects and mechanisms of ultrashort and intense pulsed electric fields on biological cells remain some unknown. Especially for picosecond pulsed electric fields (psPEF) with a high pulse repetition rate, electroporation or nanoporation effects could be induced on cell membranes and intracellular organelle membranes. In this work, the design, implementation, and experimental validation of a wideband psPEF exposure system (WPES) is reported, comprising picosecond pulser and wideband biochip, for the in vitro exposure of suspended cells to high-intensity psPEF. Excited by repetitive picosecond pulses (the duration of 200 ps and the amplitude of a few kilovolts), the proposed biochip adopts grounded coplanar waveguide (GCPW) for a wide working bandwidth, which was fabricated with 160 µm thick electrodes for uniform distribution of psPEF in the cross-section. To ensure that only psPEF is generated in the biological medium containing cells except for ionic current, this work proposes to install capillary tubes in the electrode gaps for electrical insulation and cells delivery. By electrical measurements in the time domain and frequency domain, the exposure system is adapted for local generation of extremely high-intensity psPEF with the 3 dB bandwidth up to 4.2 GHz. Furthermore, biological experiments conducted on the developed exposure system verified its capability to permeabilize biological cells under the exposure of high-intensity psPEF.

Asunto(s)

RESUMEN

Over the last few years, some novel researches in the field of medical science made a tendency to have therapy without any complications or side-effects of the disease with the aid of prognosis about the behaviors of the microtubules. Regarding this issue, the stability/instability analysis of curved microtubule-associated protein in axons with attention to different size effect parameters based on an exact continuum method is presented. The real property of the living biological cells is presented as the Kelvin-Voight viscoelastic properties. Considering length scale parameter (l/R = 0.2) in modified couple stress theory (MCST) leads to a better agreement with experimental results in comparison by other theories that in the results section is presented, in detail. Based on presented exact results, the effect of R1/R parameter on the relative frequency changes of the microtubules is hardly depended to the value of the external forced load that should be attention to this value. Another important consequence is that the influence of the microtubule curvature parameter on the relative frequency changes of the living substructure is hardly depended on the value of the time-dependent viscoelastic property, that researchers in the analysis of the microtubule should be attention to this important issue.Communicated by Ramaswamy H. Sarma.

Asunto(s)

RESUMEN

The variability in the size of red blood cells (RBCs) is an important additional diagnostic parameter for diseases, which has been established as a part of the complete blood count (CBC). The CBC can be performed using an automated flow cytometer, but it is too bulky and expensive for point-of-care testing. A miniaturized lensless imaging system is a competitive modality for a CBC, that is small and inexpensive. There are two challenges in developing a lensless imaging system for taking the CBC, which make the measurement of the RBC size very difficult: the diffraction effect and the low resolution. In this paper, the RBC radius measurement is replaced with a diffraction ring radius measurement. The diffraction ring radius is much larger than the RBC radius. This feature can improve the imaging resolution. Based on Fresnel diffraction, the relationship between the radius of RBCs and the diffraction fringes is analyzed. Finally, a complete measurement algorithm for determining the RBC size based on the lensless imaging system is given, which can be used to measure the variability in the size of RBCs. In our experiment, the maximum error is less than 6.74%.

Asunto(s)

RESUMEN

Alcohol exposure has been postulated to adversely affect the physiology and function of the red blood cells (RBCs). The global pervasiveness of alcohol abuse, causing health issues and social problems, makes it imperative to resolve the physiological effects of alcohol on RBC physiology. Alcohol consumed recreationally or otherwise almost immediately alters cell physiology in ways that is subtle and still unresolved. In this paper, we introduce a high-resolution device for quantitative electrofluidic measurement of changes in RBC volume upon alcohol exposure. We present an exhaustive calibration of our device using model cells to measure and resolve volume changes down to 0.6 fL. We find an RBC shrinkage of 5.3% at 0.125% ethanol (the legal limit in the United States) and a shrinkage of 18.5% at 0.5% ethanol (the lethal limit) exposure. Further, we also measure the time dependence of cell volume shrinkage (upon alcohol exposure) and then recovery (upon alcohol removal) to quantify shrinkage and recovery of RBC volumes. This work presents the first direct quantification of temporal and concentration-dependent changes in red blood cell volume upon ethanol exposure. Our device presents a universally applicable high-resolution and high-throughput platform to measure changes in cell physiology under native and diseased conditions.

Asunto(s)

RESUMEN

Single-connection in situ calibration using biocompatible solutions is demonstrated in single-cell sensing from 0.5 to 9 GHz. The sensing is based on quickly trapping and releasing a live cell by dielectrophoresis on a coplanar transmission line with a little protrusion in one of its ground electrodes. The same transmission line is used as the calibration standard when covered by various solutions of known permittivities. The results show that the calibration technique may be precise enough to differentiate cells of different nucleus sizes, despite the measured difference being less than 0.01 dB in the deembedded scattering parameters. With better accuracy and throughput, the calibration technique may allow broadband electrical sensing of live cells in a high-throughput cytometer.

Asunto(s)

RESUMEN

X-ray imaging of soft materials is often difficult because of the low contrast of the components. This particularly applies to frozen hydrated biological cells where the feature of interest can have a similar density to the surroundings. As a consequence, a high dose is often required to achieve the desired resolution. However, the maximum dose that a specimen can tolerate is limited by radiation damage. Results from 3D coherent diffraction imaging (CDI) of frozen hydrated specimens have given resolutions of ∼80 nm compared with the expected resolution of 10 nm predicted from theoretical considerations for identifying a protein embedded in water. Possible explanations for this include the inapplicability of the dose-fractionation theorem, the difficulty of phase determination, an overall object-size dependence on the required fluence and dose, a low contrast within the biological cell, insufficient exposure, and a variety of practical difficulties such as scattering from surrounding material. A recent article [Villaneuva-Perez et al. (2018), Optica, 5, 450-457] concluded that imaging by Compton scattering gave a large dose advantage compared with CDI because of the object-size dependence for CDI. An object-size dependence would severely limit the applicability of CDI and perhaps related coherence-based methods for structural studies. This article specifically includes the overall object size in the analysis of the fluence and dose requirements for coherent imaging in order to investigate whether there is a dependence on object size. The applicability of the dose-fractionation theorem is also discussed. The analysis is extended to absorption-based imaging and imaging by incoherent scattering (Compton) and fluorescence. This article includes analysis of the dose required for imaging specific low-contrast cellular organelles as well as for protein against water. This article concludes that for both absorption-based and coherent diffraction imaging, the dose-fractionation theorem applies and the required dose is independent of the overall size of the object. For incoherent-imaging methods such as Compton scattering, the required dose depends on the X-ray path length through the specimen. For all three types of imaging, the dependence of fluence and dose on a resolution d goes as 1/d 4 when imaging uniform-density voxels. The independence of CDI on object size means that there is no advantage for Compton scattering over coherent-based imaging methods. The most optimistic estimate of achievable resolution is 3 nm for imaging protein molecules in water/ice using lensless imaging methods in the water window. However, the attainable resolution depends on a variety of assumptions including the model for radiation damage as a function of resolution, the efficiency of any phase-retrieval process, the actual contrast of the feature of interest within the cell and the definition of resolution itself. There is insufficient observational information available regarding the most appropriate model for radiation damage in frozen hydrated biological material. It is advocated that, in order to compare theory with experiment, standard methods of reporting results covering parameters such as the feature examined (e.g. which cellular organelle), resolution, contrast, depth of the material (for 2D), estimate of noise and dose should be adopted.

RESUMEN

The rising concern over drug-resistant microorganisms has increased the need for rapid and portable detection systems. However, the traditional methods for the analysis of microorganisms can be both resource and time intensive. This contribution presents an alternative approach for the characterization of microorganisms using a microscale electrokinetic technique. The present study aims to develop and validate a library with a novel parameter referred to as the electrokinetic equilibrium condition for each strain, which will allow for fast identification of the studied bacterial and yeast cells in electrokinetic (EK) microfluidic devices. To create the library, experiments with six organisms of interest were conducted using insulator-based EK devices with circle-shaped posts. The organisms included one yeast strain, Saccharomyces cerevisiae; one salmonella strain, Salmonella enterica; two species from the same genus, Bacillus cereus and Bacillus subtilis; and two Escherichia coli strains. The results from these experiments were then analyzed with a mathematical model in COMSOL Multiphysics®, which yielded the electrokinetic equilibrium condition for each distinct strain. Lastly, to validate the applicability EK library, the COMSOL model was used to estimate the trapping conditions needed in a device with oval-shaped posts for each organism, and these values were then compared with experimentally obtained values. The results suggest the library can be used to estimate trapping voltages with a maximum relative error of 12%. While the proposed electrokinetic technique is still a novel approach and the analysis of additional microorganisms would be needed to expand the library, this contribution further supports the potential of microscale electrokinetics as a technique for the rapid and robust characterization of microbes. Graphical abstract.

Asunto(s)

RESUMEN

BACKGROUND AND OBJECTIVE: In this study, the effect of the second excitation frequency mode under different conditions on the fluid streaming and its microparticles displacement is investigated. METHODS: For this purpose, some variable parameters such as the particle diameter, microchannel aspect ratio, and applied frequency modes have been selected to study. The resulted acoustic streaming was scrutinized to understand the physics of the problem under different geometrical and input conditions. Finally, the effect of the increasing the microparticle size and aspect ratio of the microchannel, simultaneously, has been evaluated. RESULTS: The results demonstrated that increasing the microparticle size accelerates the displacement of the microparticles. On the other hand, changing the aspect ratio affects the formation of the microparticle distribution and it also changes the velocity of the microparticles due to the gradient of the second-order pressure. CONCLUSIONS: The obtained results have wide applications in the military, medical, petrochemical, and other related studies.

Asunto(s)

RESUMEN

X-ray imaging is a complementary method to electron and fluorescence microscopy for studying biological cells. In particular, scanning small-angle X-ray scattering provides overview images of whole cells in real space as well as local, high-resolution reciprocal space information, rendering it suitable to investigate subcellular nanostructures in unsliced cells. One persisting challenge in cell studies is achieving high throughput in reasonable times. To this end, a fast scanning mode is used to image hundreds of cells in a single scan. A way of dealing with the vast amount of data thus collected is suggested, including a segmentation procedure and three complementary kinds of analysis, i.e. characterization of the cell population as a whole, of single cells and of different parts of the same cell. The results show that short exposure times, which enable faster scans and reduce radiation damage, still yield information in agreement with longer exposure times.

Asunto(s)

RESUMEN

Fatigue arising from cyclic straining is a key factor in the degradation of properties of engineered materials and structures. Fatigue can also induce damage and fracture in natural biomaterials, such as bone, and in synthetic biomaterials used in implant devices. However, the mechanisms by which mechanical fatigue leads to deterioration of physical properties and contributes to the onset and progression of pathological states in biological cells have hitherto not been systematically explored. Here we present a general method that employs amplitude-modulated electrodeformation and microfluidics for characterizing mechanical fatigue in single biological cells. This method is capable of subjecting cells to static loads for prolonged periods of time or to large numbers of controlled mechanical fatigue cycles. We apply the method to measure the systematic changes in morphological and biomechanical characteristics of healthy human red blood cells (RBCs) and their membrane mechanical properties. Under constant amplitude cyclic tensile deformation, RBCs progressively lose their ability to stretch with increasing fatigue cycles. Our results further indicate that loss of deformability of RBCs during cyclic deformation is much faster than that under static deformation at the same maximum load over the same accumulated loading time. Such fatigue-induced deformability loss is more pronounced at higher amplitudes of cyclic deformation. These results uniquely establish the important role of mechanical fatigue in influencing physical properties of biological cells. They further provide insights into the accumulated membrane damage during blood circulation, paving the way for further investigations of the eventual failure of RBCs causing hemolysis in various hemolytic pathologies.

Asunto(s)

RESUMEN

With the increasing need for multi-analyte point-of-care diagnosis devices, cell impedance measurement is a promising technique for integration with other sensing modalities. In this comprehensive review, the theory underlying cell impedance sensing, including the history, complementary metal-oxide-semiconductor (CMOS) based implementations, and applications are critically assessed. Whole cell impedance sensing, also known as electric cell-substrate impedance sensing (ECIS) or electrical impedance spectroscopy (EIS), is an approach for studying and diagnosing living cells in in-vitro and in-vivo environments. The technique is popular since it is label-free, non-invasive, and low cost when compared to standard biochemical assays. CMOS cell impedance measurement systems have been focused on expanding their applications to numerous aspects of biological, environmental, and food safety applications. This paper presents and evaluates circuit topologies for whole cell impedance measurement. The presented review compares several existing CMOS designs, including the classification, measurement speed, and sensitivity of varying topologies.

Asunto(s)

RESUMEN

We propose a new deep learning approach for medical imaging that copes with the problem of a small training set, the main bottleneck of deep learning, and apply it for classification of healthy and cancer cell lines acquired by quantitative phase imaging. The proposed method, called transferring of pre-trained generative adversarial network (TOP-GAN), is hybridization between transfer learning and generative adversarial networks (GANs). Healthy cells and cancer cells of different metastatic potential have been imaged by low-coherence off-axis holography. After the acquisition, the optical path delay maps of the cells are extracted and directly used as inputs to the networks. In order to cope with the small number of classified images, we use GANs to train a large number of unclassified images from another cell type (sperm cells). After this preliminary training, we change the last layers of the network and design automatic classifiers for the correct cell type (healthy/primary cancer/metastatic cancer) with 90-99% accuracies, although small training sets of down to several images are used. These results are better in comparison to other classic methods that aim at coping with the same problem of a small training set. We believe that our approach makes the combination of holographic microscopy and deep learning networks more accessible to the medical field by enabling a rapid, automatic and accurate classification in stain-free imaging flow cytometry. Furthermore, our approach is expected to be applicable to many other medical image classification tasks, suffering from a small training set.

Asunto(s)

RESUMEN

An error in the calculation for X-ray absorption imaging has been identified in the paper by Nave (2018) [J. Synchrotron Rad. 25, 1490-1504]. The required fluence and dose in the paper are a factor of ten too low for this mode of imaging.

RESUMEN

Raman microspectroscopy is now well established as one of the most powerful analytical techniques for a diverse range of applications in physical (material) and biological sciences. Consequently, the technique provides exceptional analytical opportunities to the science and technology of biosensing due to its capability to analyze both parts of a biosensor system-biologically sensitive components, and a variety of materials and systems used in physicochemical transducers. Recent technological developments in Raman spectral imaging have brought additional possibilities in two- and three-dimensional (2D and 3D) characterization of the biosensor's constituents and their changes on a submicrometer scale in a label-free, real-time nondestructive method of detection. In this report, the essential components and features of a modern confocal Raman microscope are reviewed using the instance of Thermo Scientific DXRxi Raman imaging microscope, and examples of the potential applications of Raman microscopy and imaging for constituents of biosensors are presented.

Asunto(s)