Label-free Methods Research Articles

Picofluidic Electro-Osmosis Measurement of Cell Membrane Mechanical Properties.

Cells connect with their internal and external environments through plasma membranes, and the mechanical properties of cell membranes govern numerous biological events. Membrane detection techniques such as optical or magnetic tweezers have revealed mechanical strength by membrane-anchored modifications, but it remains challenging to develop label-free methods to reduce the influence of exogenous interference. Here picofluidic electro-osmosis measurement (PEOM), which enables direct and efficient sensing of cell membrane mechanical properties by using a glass nanopipette without labeling, is presented. By generating a picoliter electroosmotic fluid at the nanopipette tip, periodic cell membrane vibration modes are observed from current traces, which carry information on membrane mechanical properties to indicate its biological state. Based on characteristic peaks in the frequency domain, a theoretical framework to describe the vibration modes, which contains two ideal spring vibrator models corresponding to stretching and bending vibrations of cell membrane respectively, is developed. Notably, the PEOM strategy represents a label-free approach to reveal the mechanical properties of living cell membranes from two dimensions, which is completely different from other methods. Additionally, the exciting potential of PEOM is demonstrated for label-free observation of membrane mechanical property changes during different bioprocesses, including cytoskeletal alteration, membrane tension change, and mechanical polarization.

Discovery of peptide quality markers for quality control and identification of toad venom using LC-MS/MS and label-free methods

Discovery of peptide quality markers for quality control and identification of toad venom using LC-MS/MS and label-free methods

Two-Step Acoustic Cell Separation Based on Cell Size and Acoustic Impedance─toward Isolation of Viable Circulating Tumor Cells.

Isolation and characterization of circulating tumor cells (CTCs) present a noninvasive alternative to monitor disease progression in individual patients. However, the heterogeneous lineage specificity of CTCs makes it difficult to isolate and identify possible CTCs by a liquid biopsy. Better label-free methods for the isolation of viable CTCs are needed. Our solution is a combined approach that is inherently epitope independent. Cells are separated by size-sensitive acoustophoresis using an ultrasonic standing wave field, followed by size-insensitive, acoustic barrier-medium focusing, which enables the enrichment of viable cancer cells in blood. With standard acoustophoresis in homogeneous medium, lymphocytes and monocytes were efficiently removed, while removal of granulocytes from the target MCF7 breast cancer cells was not possible due to overlapping acoustic migration velocities for viable cells. Remaining granulocytes were removed by a second separation step with an acoustic impedance barrier-medium selectively blocking the transport of MCF7 cells to generate a clean cancer cell fraction. For two series of 500 mL samples containing 5 × 105 white blood cells, spiked with 2 × 104 or 1 × 103 MCF7 cells, the recovery of MCF7 cells was 77.3% with a 99.9% depletion of white blood cells in the final cancer cell fraction. The most abundant contaminating cell type was granulocytes (85.9% of remaining cells). Nearly all lymphocytes (99.996%) and monocytes (99.995%) were depleted. A two-step acoustic cell separation based on cell size and acoustic impedance is well suited to generate a purified cancer cell fraction as a preparatory step for downstream single-cell analysis.

Deciphering Cancer Complexity: Integrative Proteogenomics and Proteomics Approaches for Biomarker Discovery.

Proteomics has revolutionized the field of cancer biology because the use of a large number of in vivo (SILAC), in vitro (iTRAQ, ICAT, TMT, stable-isotope Dimethyl, and 18O) labeling techniques or label-free methods (spectral counting or peak intensities) coupled with mass spectrometry enables us to profile and identify dysregulated proteins in diseases such as cancer. These proteome and genome studies have led to many challenges, such as the lack of consistency or correlation between copy numbers, RNA, and protein-level data. This review covers solely mass spectrometry-based approaches used for cancer biomarker discovery. It also touches on the emerging role of oncoproteogenomics or proteogenomics in cancer biomarker discovery and how this new area is attracting the integration of genomics and proteomics areas to address some of the important questions to help impinge on the biology and pathophysiology of different malignancies to make these mass spectrometry-based studies more realistic and relevant to clinical settings.

Dissociation Constant (Kd) Measurement for Small-Molecule Binding Aptamers: Homogeneous Assay Methods and Critical Evaluations.

Since 1990, numerous aptamers have been isolated and discovered for use in various analytical, biomedical, and environmental applications. This trend continues to date. A critical step in the characterization of aptamer binding is to measure its binding affinity toward both target and non-target molecules. Dissociation constant (Kd) is the most commonly used value in characterizing aptamer binding. In this article, homogenous assays are reviewed for aptamers that can bind small-molecule targets. The reviewed methods include label-free methods, such as isothermal titration calorimetry, intrinsic fluorescence of target molecules, DNA staining dyes, and nuclease digestion assays, and labeled methods, such as the strand displacement reaction. Some methods are not recommended, such as those based on the aggregation of gold nanoparticles and the desorption of fluorophore-labeled DNA from nanomaterials. The difference between the measured apparent Kd and the true Kd of aptamer binding is stressed. In addition, avoiding the titration regime and paying attention to the time required to reach equilibrium are discussed. Finally, it is important to include mutated non-binding sequences as controls.

Measurable morphological features of single circulating tumor cells in selected solid tumors-A pilot study.

Liquid biopsies developed into a range of sensitive technologies aiming to analyze for example, circulating tumor cells (CTCs) in peripheral blood, which significantly deepens understanding of the metastatic process. Nevertheless, examination of CTCs is mostly limited to their enumeration and usually only 2-3 markers-based phenotyping, not offering yet sufficient insight into their biology. In contrast, quantitative analysis of their morphological details might extend our knowledge about dissemination and even improve CTC isolation or label-free identification methods dependent on their physical features such as size, and deformability. Current study was conducted to describe CTCs' and their size, shape, presence of protrusions, and micronuclei across various types of cancers (lung, n = 29; ovarian, n = 24, breast, n = 54; and prostate, n = 33). Epithelial (pan-keratins), mesenchymal (vimentin), and two exclusion markers were used to identify CTCs and classify them into four epithelial and epithelial-mesenchymal transition-related phenotypes using standardized and throughput method, imaging flow cytometry. The morphological characteristics of CTCs, including their nuclei, such as circularity, the maximum, and minimum diagonal values were determined using an open-source software QuPath. On average, detected CTCs (n = 1156) were larger, and more irregular in shape compared to leukocytes/endothelial cells (n = 400). Epithelial and mesenchymal CTCs had the largest (median = 18.2 μm) and the smallest diameter (median = 10.4 μm), respectively. In terms of cancer-specific variations, the largest CTCs were identified in lung cancer, whereas the smallest-in prostate and breast cancers. Epithelial CTCs and those negative for both epithelial and mesenchymal markers exhibited the highest degree of elongation, whereas mesenchymal CTCs were the most irregular in shape. Protrusions and micronuclei were observed extremely rarely within CTCs of breast and prostate cancer (0.6%-0.8% of CTCs). Micronuclei were observed only in epithelial and epithelial-mesenchymal CTCs. This study underscores the significant variability in the morphological features of CTCs in relation to their phenotypic classification or even the particular organ of origin, potentially influencing for example, size-dependent CTC isolation methods. It demonstrates for the first time the morphological measurements of CTCs undergoing epithelial-mesenchymal transition, and some specific morphological details (i.e., protrusions, micronuclei) within CTCs in general.

Developments in Localized Surface Plasmon Resonance

AbstractLocalized surface plasmon resonance (LSPR) is a nanoscale phenomenon associated with noble metal nanostructures that has long been studied and has gained considerable interest in recent years. These resonances produce sharp spectral absorption and scattering peaks, along with strong electromagnetic near-field enhancements. Over the past decade, advancements in the fabrication of noble metal nanostructures have propelled significant developments in various scientific and technological aspects of LSPR. One notable application is the detection of molecular interactions near the nanoparticle surface, observable through shifts in the LSPR spectral peak. This document provides an overview of this sensing strategy. Given the broad and expanding scope of this topic, it is impossible to cover every aspect comprehensively in this review. However, we aim to outline major research efforts within the field and review a diverse array of relevant literature. We will provide a detailed summary of the physical principles underlying LSPR sensing and address some existing inconsistencies in the nomenclature used. Our discussion will primarily focus on LSPR sensors that employ metal nanoparticles, rather than on those utilizing extended, fabricated structures. We will concentrate on sensors where LSPR acts as the primary mode of signal transduction, excluding hybrid strategies like those combining LSPR with fluorescence. Additionally, our examination of biological LSPR sensors will largely pertain to label-free detection methods, rather than those that use metal nanoparticles as labels or as means to enhance the efficacy of a label. In the subsequent section of this review, we delve into the analytical theory underpinning LSPR, exploring its physical origins and its dependency on the material properties of noble metals and the surrounding refractive index. We will discuss the behavior of both spherical and spheroidal particles and elaborate on how the LSPR response varies with particle aspect ratio. Further, we detail the fundamentals of nanoparticle-based LSPR sensing. This includes an exploration of single-particle and ensemble measurements and a comparative analysis of scattering, absorption, and extinction phenomena. The discussion will extend to how these principles are applied in practical sensing scenarios, highlighting the key experimental approaches and measurement techniques.

Label-free and TMT-labeled Proteomics Methods to Compare Differences on Normal and Attached Livers of Glyptosternum maculatum

Background: The aim of this study is to compare the molecular differences between the normal liver (NG) and attached liver (WG) of Glyptosternum maculatum (G. maculatum) based on label-free and TMT-labeled proteomics data. It provides a theoretical basis for the adaptation of G. maculatum in the plateau area with low temperature and low oxygen Methods: The differentially expressed proteins (DEPs) of label-free and TMT-labeled proteomics data were analyzed by Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, and protein structural domain (PSD) enrichment, and protein network interactions were constructed to reveal protein characteristics. Results: detected a total of 643 and 107 DEPs; 7 common DEPs (co-DEPs) were selected through PPI network information. Higher expression of evm.model.chr5.73, evm.model.chr18.610, evm.model.chr10.1201, and HIF-1 signaling pathway were associated with a hypoxic environment. evm.model.chr15.573, evm.model .chr5.320, evm.- model.chr6.360, and evm.model.chr18.610 were associated with WG production. Significant expression of Ubiquitin-like protein 4A and Heat shock 70 kDa protein protects liver cells from injury in a hypothermic and hypoxic environment. Conclusion: In terms of the number of DEPs, the labelling-free method is superior to the TMT-labeled method. The TMT-labeled method is better in terms of repeatability and accuracy. The myosin11 not only responds to low temperature and low oxygen but may also lead to WG production through Tight junctions. The NG may be more sensitive than WG in stress response to cancer and viral infections.

Deep learning classification method for boar sperm morphology analysis.

Boar semen quality emphasizes three major criteria: sperm concentration, motility, and morphology. Methods to analyze concentration and motility quickly and objectively readily exist, but few exist for analyzing morphology outside of subjective manual counting. Other vital factors for fertilization, like acrosome health, lack efficient detection methods due to limitations in detection by the human eye and costly biomarker analysis, which is rarely used in semen diagnostics. To overcome these challenges, we propose a novel approach integrating deep-learning technology with high-throughput image-based flow cytometry (IBFC) for objective and accurate analysis of both morphology and label-free acrosome health of thousands of individual spermatozoa at once, as opposed to manually counting on a microscope slide. Images of 10,000 spermatozoa were captured using an IBFC and manually annotated based on the primary morphological defect or acrosome health status for the training of the convolutional neural network (CNN). The CNN used these images to train and then applied that training to unannotated images to predict the model accuracy. Using the CNNs, high F1 scores of 96.73%, 98.55%, and 99.31% for 20x, 40x, and 60x magnifications, respectively, for morphological classification were attained. Additionally, the model demonstrates an F1 score of 99.8% in detecting subtle acrosome health variations at the 60x magnification. We have established an integrated approach to rapidly collect and classify morphological defects and acrosome health status, without the use of manual counting or biomarker labeling. Our study underscores the potential of artificial intelligence in semen diagnostics, reducing technician variability, streamlining assays, and facilitating the development of additional label-free detection methods. This innovative approach addresses the barriers hindering biomarker adoption in semen analysis, offering a promising avenue for enhancing reproductive health assessments.

Radiobiological Applications of Vibrational Spectroscopy: A Review of Analyses of Ionising Radiation Effects in Biology and Medicine

Vibrational spectroscopic techniques, such as Fourier transform infrared (FTIR) absorption and Raman spectroscopy (RS), offer unique and detailed biochemical fingerprints by detecting specific molecular vibrations within samples. These techniques provide profound insights into the molecular alterations induced by ionising radiation, which are both complex and multifaceted. This paper reviews the application of rapid and label-free vibrational spectroscopic methods for assessing biological radiation responses. These assessments span from early compartmentalised models such as DNA, lipid membranes, and vesicles to comprehensive evaluations in various living biological models, including tissues, cells, and organisms of diverse origins. The review also discusses future perspectives, highlighting how the field is overcoming methodological limitations. RS and FTIR have demonstrated significant potential in detecting radiation-induced biomolecular alternations, which may facilitate the identification of radiation exposure spectral biomarkers/profiles.

Site-specific N-glycan alterations on haptoglobin as potential biomarkers for distinguishing intrahepatic cholangiocarcinoma from hepatocellular carcinoma

Intrahepatic cholangiocellular carcinoma (ICC) is a challenging malignancy marked by subtle early symptoms and a high mortality rate, making effective diagnostic markers crucial for early detection and improved patient outcomes. Currently, the conventional diagnosis of ICC is not easily distinguishable from Hepatocellular Carcinoma (HCC) and lacks highly specific and sensitive diagnostic markers. Protein glycosylation, pivotal in biological processes, shows promise for cancer biomarkers due to its association with disease progression. This study aims to develop a novel biomarker discovery framework for ICC utilizing site-specific quantitative N-glycoproteomics to overcome the limitations of existing diagnostic approaches. Employing a tandem mass tag (TMT)-based quantitative analysis, we profiled serum glycoproteins from ICC, HCC, and control cohorts at site-specific glycosylation level. The identified markers underwent further validation in an independent cohort using label-free quantitative methods. Ultimately, we identified five site-specific N-glycans on haptoglobin (HP) as potential biomarkers (AUC > 0.9) for distinguishing ICC from HCC. This finding represents a considerable advance over traditional biomarkers, highlighting the significance of protein glycosylation alterations in ICC pathogenesis. This research, therefore, sets a new precedent for biomarker discovery in ICC, with potential applications in other cancers characterized by glycosylation abnormalities.

Statistical estimation theory detection limits for label-free imaging.

The emergence of label-free microscopy techniques has significantly improved our ability to precisely characterize biochemical targets, enabling non-invasive visualization of cellular organelles and tissue organization. However, understanding each label-free method with respect to the specific benefits, drawbacks, and varied sensitivities under measurement conditions across different types of specimens remains a challenge. We link all of these disparate label-free optical interactions together and compare the detection sensitivity within the framework of statistical estimation theory. To achieve this goal, we introduce a comprehensive unified framework for evaluating the bounds for signal detection with label-free microscopy methods, including second-harmonic generation, third-harmonic generation, coherent anti-Stokes Raman scattering, coherent Stokes Raman scattering, stimulated Raman loss, stimulated Raman gain, stimulated emission, impulsive stimulated Raman scattering, transient absorption, and photothermal effect. A general model for signal generation induced by optical scattering is developed. Based on this model, the information obtained is quantitatively analyzed using Fisher information, and the fundamental constraints on estimation precision are evaluated through the Cramér-Rao lower bound, offering guidance for optimal experimental design and interpretation. We provide valuable insights for researchers seeking to leverage label-free techniques for non-invasive imaging applications for biomedical research and clinical practice.

Nanotechnology-Enabled PCR with Tunable Energy Dynamics.

This Perspective elucidates the transformative impacts of advanced nanotechnology and dynamic energy systems on the polymer chain reaction (PCR), a cornerstone technique in biomedical research and diagnostic applications. Since its invention, the optimization of PCR-specifically its efficiency, specificity, cycling rate, and detection sensitivity-has been a focal point of scientific exploration. Our analysis spans the modulation of PCR from both material and energetic perspectives, emphasizing the intricate interplay between PCR components and externally added entities such as molecules, nanoparticles (NPs), and optical microcavities. We begin with a foundational overview of PCR, detailing the basic principles of PCR modulation through molecular additives to highlight material-level interactions. Then, we delve into how NPs, with their diverse material and surface properties, influence PCR through interface interactions and hydrothermal conduction, drawing parallels to molecular behaviors. Additionally, this Perspective ventures into the energetic regulation of PCR, examining the roles of electromagnetic radiation and optical resonators. We underscore the advanced capabilities of optical technologies in PCR regulation, characterized by their ultrafast, residue-free, and noninvasive nature, alongside label-free detection methods. Notably, optical resonators present a pioneering approach to control PCR processes even in the absence of light, targeting the often-overlooked water component in PCR. By integrating discussions on photocaging and vibrational strong coupling, this review presents innovative methods for the precise regulation of PCR processes, envisioning a new era of PCR technology that enhances both research and clinical diagnostics. The synergy between nanotechnological enhancements and energy dynamics not only enriches our understanding of PCR but also opens new avenues for developing rapid, accurate, and efficient PCR systems. We hope that this Perspective will inspire further innovations in PCR technology and guide the development of next-generation clinical detection instruments.

Surpassing the Diffraction Limit in Label-Free Optical Microscopy.

Super-resolution optical microscopy has enhanced our ability to visualize biological structures on the nanoscale. Fluorescence-based techniques are today irreplaceable in exploring the structure and dynamics of biological matter with high specificity and resolution. However, the fluorescence labeling concept narrows the range of observed interactions and fundamentally limits the spatiotemporal resolution. In contrast, emerging label-free imaging methods are not inherently limited by speed and have the potential to capture the entirety of complex biological processes and dynamics. While pushing a complex unlabeled microscopy image beyond the diffraction limit to single-molecule resolution and capturing dynamic processes at biomolecular time scales is widely regarded as unachievable, recent experimental strides suggest that elements of this vision might be already in place. These techniques derive signals directly from the sample using inherent optical phenomena, such as elastic and inelastic scattering, thereby enabling the measurement of additional properties, such as molecular mass, orientation, or chemical composition. This perspective aims to identify the cornerstones of future label-free super-resolution imaging techniques, discuss their practical applications and theoretical challenges, and explore directions that promise to enhance our understanding of complex biological systems through innovative optical advancements. Drawing on both traditional and emerging techniques, label-free super-resolution microscopy is evolving to offer detailed and dynamic imaging of living cells, surpassing the capabilities of conventional methods for visualizing biological complexities without the use of labels.

Enzyme-free fluorescent DNA detection based on nucleic acid-templated click reaction via controllable synthesis of Cu2O as heterogeneous nanocatalyst

In the field of nucleic acid amplification assays, developing enzyme-free, easy-to-use, and highly sensitive amplification approaches remains a challenge. In this work, we synthesized a heterogeneous Cu2O nanocatalyst (hnCu2O) with different particle sizes and shapes, which was used for developing enzyme- and label-free nucleic acid amplification methods based on the nucleic acid-templated azide–alkyne cycloaddition (AAC) reaction catalyzed by hnCu2O. The hnCu2O exhibited size- and shape-dependent catalytic activity, with smaller sizes and spherical-like shapes exhibiting superior activity. Spherical-like hnCu2O (61 ± 8 nm) not only achieved a ligation yield of up to 84.2 ± 3.9 % in 3 min but also exhibited faster kinetics in the nucleic acid-templated hnCu2O-catalyzed AAC reaction, with a high reaction rate of 0.65 min−1 and a half-life of 1.07 ± 0.09 min. Based on this result, we developed nucleic acid-templated click ligation linear amplification reaction (NA-CLLAR) and nucleic acid-templated click ligation exponential amplification reaction (NA-CLEAR) approach. By combining the recognition (complementary to the target sequence) and signal output (split G-quadruplex sequence) elements into a DNA probe, the NA-CLLAR and NA-CLEAR fluorescence assays achieved highly specific detection of target nucleic acids, with a detection limit of 2.8 aM based on G-quadruplex-enhanced fluorescence. This work is a valuable reference and will inspire researchers to design enzyme-free nucleic acid signal amplification strategies by developing different types of Cu(I) catalysts with improved catalytic activity.

Underlying mechanism of Dendrobium huoshanense resistance to lead stress using the quantitative proteomics method

Lead affects photosynthesis and growth and has serious toxic effects on plants. Here, the differential expressed proteins (DEPs) in D. huoshanense were investigated under different applications of lead acetate solutions. Using label-free quantitative proteomics methods, more than 12,000 peptides and 2,449 proteins were identified. GO and KEGG functional annotations show that these differential proteins mainly participate in carbohydrate metabolism, energy metabolism, amino acid metabolism, translation, protein folding, sorting, and degradation, as well as oxidation and reduction processes. A total of 636 DEPs were identified, and lead could induce the expression of most proteins. KEGG enrichment analysis suggested that proteins involved in processes such as homologous recombination, vitamin B6 metabolism, flavonoid biosynthesis, cellular component organisation or biogenesis, and biological regulation were significantly enriched. Nearly 40 proteins are involved in DNA replication and repair, RNA synthesis, transport, and splicing. The effect of lead stress on D. huoshanense may be achieved through photosynthesis, oxidative phosphorylation, and the production of excess antioxidant substances. The expression of 9 photosynthesis-related proteins and 12 oxidative phosphorylation-related proteins was up-regulated after lead stress. Furthermore, a total of 3 SOD, 12 POD, 3 CAT, and 7 ascorbate-related metabolic enzymes were identified. Under lead stress, almost all key enzymes involved in the synthesis of antioxidant substances are up-regulated, which may facilitate the scavenging of oxygen-free radical scavenging. The expression levels of some key enzymes involved in sugar and glycoside synthesis, the phenylpropanoid synthesis pathway, and the terpene synthesis pathway also increased. More than 30 proteins involved in heavy metal transport were also identified. Expression profiling revealed a significant rise in the expression of the ABC-type multidrug resistance transporter, copper chaperone, and P-type ATPase with exposure to lead stress. Our findings lay the basis for research on the response and resistance of D. huoshanense to heavy metal stress.

Electrochemical Label-free Methods for Ultrasensitive Multiplex Protein Profiling of Infectious Diseases.

Electrochemical detection methods are the more appropriate detection methods when it comes to the sensitive and specific determination of biomarkers. Biomarkers are the biological targets for disease diagnosis and monitoring. This review focuses on recent advances in label-free detection of biomarkers for infectious disease diagnosis. The current state of the art for rapid detection of infectious diseases and their clinical applications and challenges were discussed. Label-free electroanalytical methods are probably the most promising means to achieve this. We are currently in the early stages of the emerging technology of using label-free electrochemistry of proteins to develop biosensors. To date, antibody-based biosensors have been intensively developed, although many improvements in reproducibility and sensitivity are still needed. Moreover, there is no doubt that a growing number of aptamers and hopefully label-free biosensors based on nanomaterials will soon be used for disease diagnosis and therapy monitoring. And also here in this review article, we have discussed recent developments in the diagnosis of bacterial and viral infections, as well as the current status of the use of label-free electrochemical methods for monitoring inflammatory diseases.

Label-free evanescent imaging of cellular heterogeneity in membrane protein binding kinetics

Quantifying cellular heterogeneity of membrane protein binding kinetics is challenging but important for exploring drug resistance and screening drugs. Label-free analysis methods have emerged as promising tools for in situ binding kinetics analysis, but they have not been used for high throughput single cell analysis in live cells. Here we show that this is possible with Evanescent Scattering Microscopy (ESM). The ESM permits analyzing the kinetics of ligand binding onto membrane proteins in individual fixed and live cells, and provides a throughput of ∼200 cells in a single measurement with a period of ∼7 minutes. The statistical analysis further shows that the dissociation rate constant dominates the heterogeneity of cell responses to ligand binding, providing evidence for a long-standing hypothesis that the drug-target residence time may play a critical role in drug treatment. In addition, the ESM reveals that under some conditions the cells have responses to drug binding at the single cell level, whereas the ensemble measurements may average out the individual differences and present false negative results. We anticipate that the new evanescent imaging method will provide a powerful tool to quantify the functions of cellular proteins, especially their cell-to-cell heterogeneity that can provide fuel for drug resistance.

ISCAT microscopy and particle tracking with tailored spatial coherence

Interferometric scattering (iSCAT) microscopy has demonstrated unparalleled performance among label-free optical methods for detecting and imaging isolated nanoparticles and molecules. However, when imaging complex structures such as biological cells, the superposition of the scattering fields from different locations of the sample leads to a speckle-like background, posing a significant challenge in deciphering fine features. Here, we show that by controlling the spatial coherence of the illumination, one can eliminate the spurious speckle without sacrificing sensitivity. We demonstrate this approach by positioning a rotating diffuser coupled with an adjustable lens and an iris in the illumination path. We report on imaging at a high frame rate of 25 kHz and across a large field of view of 100µm×100µm, while maintaining diffraction-limited resolution. We showcase the advantages of these features by three-dimensional (3D) tracking over 1000 vesicles in a single COS-7 cell and by imaging the dynamics of the endoplasmic reticulum (ER) network. Our approach opens the door to the combination of label-free imaging, sensitive detection, and 3D high-speed tracking using wide-field iSCAT microscopy.

Reproducibility analysis of bioimpedance-based self-developed live cell assays

Bioimpedance spectrum (BIS) measurements have a great future in in vitro experiments, meeting all the requirements for non-destructive and label-free methods. Nevertheless, a real basic research can provide the necessary milestones to achieve the success of the method. In this paper a self-developed technology-based approach for in vitro assays is proposed. Authors invented a special graphene-based measuring plate in order to assess the high sensitivity and reproducibility of introduced technique. The design of the self-produced BIS plates maximizes the detection capacity of qualitative changes in cell culture and it is robust against physical effects and artifacts. The plates do not influence the viability and proliferation, however the results are robust, stable and reproducible regardless of when and where the experiments are carried out. In this study, physiological saline concentrations, two cancer and stem cell lines were utilized. All the results were statistically tested and confirmed. The findings of the assays show, that the introduced BIS technology is appropriate to be used in vitro experiments with high efficacy. The experimental results demonstrate high correlation values across the replicates, and the model parameters suggested that the characteristic differences among the various cell lines can be detected using appropriate hypothesis tests.