Monitoring the molecular composition of live cells exposed to electric pulses via label-free optical methods

Antibiotic resistance is creating enormous attention on the development of new antibiotic-free therapy strategies for bacterial diseases. Mesenchymal stromal stem cells (MSCs) are the most promising candidates in current clinical trials and included in several cell-therapy protocols. Together with the well-known immunomodulatory and regenerative potential of the MSC secretome, these cells have shown direct and indirect anti-bacterial effects. However, the low reproducibility and standardization of MSCs from different sources are the current limitations prior to the purification of cell-free secreted antimicrobial peptides and exosomes. In order to improve MSC characterization, novel label-free functional tests, evaluating the biophysical properties of the cells, will be advantageous for their cell profiling, population sorting, and quality control. We discuss the potential of emerging microfluidic technologies providing new insights into density, shape, and size of live cells, starting from heterogeneous or 3D cultured samples. The prospective application of these technologies to studying MSC populations may contribute to developing new biopharmaceutical strategies with a view to naturally overcoming bacterial defense mechanisms.

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Non-Destructive Label-Free Monitoring of Drug Intake in Live Cells using ATR FT-IR Spectroscopy

Biophysical Journal ◽

10.1016/j.bpj.2014.11.2696 ◽

2015 ◽

Vol 108 (2) ◽

pp. 493a ◽

Cited By ~ 2

Author(s):

Pedro L. Fale ◽

K.L. Andrew Chan

Keyword(s):

Ir Spectroscopy ◽

Drug Intake ◽

Live Cells ◽

Label Free ◽

Ft Ir ◽

Non Destructive ◽

Ft Ir Spectroscopy

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Nanoscale chromatin imaging and analysis platform bridges 4D chromatin organization with molecular function

Science Advances ◽

10.1126/sciadv.abe4310 ◽

2021 ◽

Vol 7 (1) ◽

pp. eabe4310

Author(s):

Yue Li ◽

Adam Eshein ◽

Ranya K.A. Virk ◽

Aya Eid ◽

Wenli Wu ◽

...

Keyword(s):

Electron Tomography ◽

Super Resolution ◽

Population Level ◽

Average Diameter ◽

Live Cells ◽

Molecular Function ◽

Label Free ◽

Multiple Length Scales ◽

Chromatin Packing ◽

Analysis Platform

Extending across multiple length scales, dynamic chromatin structure is linked to transcription through the regulation of genome organization. However, no individual technique can fully elucidate this structure and its relation to molecular function at all length and time scales at both a single-cell level and a population level. Here, we present a multitechnique nanoscale chromatin imaging and analysis (nano-ChIA) platform that consolidates electron tomography of the primary chromatin fiber, optical super-resolution imaging of transcription processes, and label-free nano-sensing of chromatin packing and its dynamics in live cells. Using nano-ChIA, we observed that chromatin is localized into spatially separable packing domains, with an average diameter of around 200 nanometers, sub-megabase genomic size, and an internal fractal structure. The chromatin packing behavior of these domains exhibits a complex bidirectional relationship with active gene transcription. Furthermore, we found that properties of PDs are correlated among progenitor and progeny cells across cell division.

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Volumetric registration of magnetic nanoparticles for optimization of quantitative immunochromatographic assays for detection of small molecules

EPJ Web of Conferences ◽

10.1051/epjconf/201818510006 ◽

2018 ◽

Vol 185 ◽

pp. 10006 ◽

Cited By ~ 2

Author(s):

Natalia V. Guteneva ◽

Sergey L. Znoyko ◽

Alexey V. Orlov ◽

Maxim P. Nikitin ◽

Petr I. Nikitin

Keyword(s):

Magnetic Nanoparticles ◽

Small Molecules ◽

Optical Methods ◽

Magnetic Method ◽

Label Free ◽

Spectral Correlation ◽

Entire Volume ◽

Specificity And Sensitivity ◽

Particle Quantification

Precise quantitative and highly sensitive detection of small molecules (haptens) is highly demanded in medicine, food quality control, in vitro diagnostics, criminalistics, environmental monitoring, etc. In the present work, the magnetic method of particle quantification and the optical methods of spectral correlation and spectral phase interferometry complement each other for optimization of a quantitative assay for measuring concentrations of small molecules. The assay employs magnetic nanoparticles as labels in rapid immunochromatographic format. The approach was demonstrated with fluorescein as a model molecule. The interferometric label-free biosensors were employed for selection of optimal reagents that produced high specificity and sensitivity. The method of magnetic particle quantification counted the magnetic labels over the entire volume of the immunochromatographic membrane to provide their distribution along the test strip. Such distribution was used for optimization of such parameters as concentrations of the used reagents and of antibody immobilized on the labels, amount of the labels and conjugates of haptens with protein carriers to realize the advanced quantitative immunochromatographic assay.

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Label-Free Digital Holo-tomographic Microscopy Reveals Virus-Induced Cytopathic Effects in Live Cells

mSphere ◽

10.1128/mspheredirect.00599-18 ◽

2018 ◽

Vol 3 (6) ◽

Cited By ~ 5

Author(s):

Artur Yakimovich ◽

Robert Witte ◽

Vardan Andriasyan ◽

Fanny Georgi ◽

Urs F. Greber

Keyword(s):

Refractive Index ◽

Vaccinia Virus ◽

Virus Type ◽

Live Cells ◽

Refractive Index Gradient ◽

Label Free ◽

Virus Infections ◽

Cytopathic Effects ◽

Infected Cells ◽

Index Gradient

ABSTRACTCytopathic effects (CPEs) are a hallmark of infections. CPEs are difficult to observe due to phototoxicity from classical light microscopy. We report distinct patterns of virus infections in live cells using digital holo-tomographic microscopy (DHTM). DHTM is label-free and records the phase shift of low-energy light passing through the specimen on a transparent surface with minimal perturbation. DHTM measures the refractive index (RI) and computes the refractive index gradient (RIG), unveiling optical heterogeneity in cells. We find that vaccinia virus (VACV), herpes simplex virus (HSV), and rhinovirus (RV) infections progressively and distinctly increased RIG. VACV infection, but not HSV and RV infections, induced oscillations of cell volume, while all three viruses altered cytoplasmic membrane dynamics and induced apoptotic features akin to those caused by the chemical compound staurosporine. In sum, we introduce DHTM for quantitative label-free microscopy in infection research and uncover virus type-specific changes and CPE in living cells with minimal interference.IMPORTANCEThis study introduces label-free digital holo-tomographic microscopy (DHTM) and refractive index gradient (RIG) measurements of live, virus-infected cells. We use DHTM to describe virus type-specific cytopathic effects, including cyclic volume changes of vaccinia virus infections, and cytoplasmic condensations in herpesvirus and rhinovirus infections, distinct from apoptotic cells. This work shows for the first time that DHTM is suitable to observe virus-infected cells and distinguishes virus type-specific signatures under noninvasive conditions. It provides a basis for future studies, where correlative fluorescence microscopy of cell and virus structures annotate distinct RIG values derived from DHTM.

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Nanoscale Chromatin Imaging and Analysis (nano-ChIA) platform bridges 4-D chromatin organization with molecular function

10.1101/2020.01.26.920363 ◽

2020 ◽

Author(s):

Yue Li ◽

Adam Eshein ◽

Ranya K.A. Virk ◽

Aya Eid ◽

Wenli Wu ◽

...

Keyword(s):

Chromatin Structure ◽

Electron Tomography ◽

Population Level ◽

Average Diameter ◽

Chromatin Organization ◽

Live Cells ◽

Molecular Function ◽

Label Free ◽

A Cell ◽

Chromatin Packing

AbstractIn eukaryotic cells, chromatin structure is linked to transcription processes through the regulation of genome organization. Extending across multiple length-scales - from the nucleosome to higher-order three-dimensional structures - chromatin is a dynamic system which evolves throughout the lifetime of a cell. However, no individual technique can fully elucidate the behavior of chromatin organization and its relation to molecular function at all length- and timescales at both a single-cell and a cell population level. Herein, we present a multi-technique nanoscale Chromatin Imaging and Analysis (nano-ChIA) platform that bridges electron tomography and optical superresolution imaging of chromatin conformation and transcriptional processes, with resolution down to the level of individual nucleosomes, with high-throughput, label-free analysis of chromatin packing and its dynamics in live cells. Utilizing nano-ChIA, we observed that chromatin is localized into spatially separable packing domains, with an average diameter of around 200 nm, sub-Mb genomic size, and an internal fractal structure. The chromatin packing behavior of these domains is directly influenced by active gene transcription. Furthermore, we demonstrated that the chromatin packing domain structure is correlated among progenitor cells and all their progeny, indicating that the organization of chromatin into fractal packing domains is heritable across cell division. Further studies employing the nano-ChIA platform have the potential to provide a more coherent picture of chromatin structure and its relation to molecular function.

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Label-free 3D refractive index interferometric tomography of live cells with full angular coverage

Label-free Biomedical Imaging and Sensing (LBIS) 2021 ◽

10.1117/12.2578229 ◽

2021 ◽

Author(s):

Matan Dudaie ◽

Itay Barnea ◽

Natan T. Shaked

Keyword(s):

Refractive Index ◽

Live Cells ◽

Label Free

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Monitoring the effects of chemical stimuli on live cells with metasurface-enhanced infrared reflection spectroscopy

10.1101/2021.06.30.450584 ◽

2021 ◽

Author(s):

Steven H. Huang ◽

Jiaruo Li ◽

Zhiyuan Fan ◽

Robert Delgado ◽

Gennady Shvets

Keyword(s):

Intracellular Signaling ◽

Strong Field ◽

Detection System ◽

Field Enhancement ◽

Chemical Imaging ◽

Live Cells ◽

Label Free ◽

Reflection Spectroscopy ◽

Infrared Reflection ◽

Infrared Reflection Spectroscopy

Infrared spectroscopy has found wide applications in the analysis of biological materials. A more recent development is the use of engineered nanostructures, or plasmonic metasurfaces, as substrates for metasurface-enhanced infrared reflection spectroscopy (MEIRS). Here, we demonstrate that strong field enhancement from plasmonic metasurfaces enables the use of MEIRS as a highly informative analytic technique for real-time monitoring of cells. By exposing live cells cultured on a plasmonic metasurface to chemical compounds, we show that MEIRS can be used as a label-free phenotypic assay for detecting multiple cellular responses to external stimuli: changes in cell morphology, adhesion, lipid composition of the cellular membrane, as well as intracellular signaling. Using a focal plane array detection system, we show that MEIRS also enables spectro-chemical imaging at the single-cell level. The described metasurface-based all-optical sensor opens the way to a scalable, high-throughput spectroscopic assay for live cells.

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Tracking the Formation and Degradation of Fatty-Acid-Accumulated Mitochondria Using Label-Free Chemical Imaging

10.21203/rs.3.rs-119989/v1 ◽

2020 ◽

Author(s):

Chi Zhang ◽

Stephen Boppart

Keyword(s):

Fatty Acid ◽

Structural Features ◽

Chemical Imaging ◽

Live Cells ◽

Label Free ◽

Dynamic Changes ◽

Regulatory Processes ◽

Transport Chain ◽

Anti Stokes ◽

First Time

Abstract The mitochondrion is one of the key organelles for maintaining cellular homeostasis. External environmental stimuli and internal regulatory processes alter the metabolism and functions of mitochondria. To understand these activities of mitochondria, it is critical to probe the key metabolic molecules inside these organelles. In this study, we used label-free chemical imaging modalities including coherent anti-Stokes Raman scattering and multiphoton-excited autofluorescence to study the mitochondrial activities in living cancer cells. We found that hypothermia exposure tends to induce fatty-acid (FA) accumulation in some mitochondria of MIAPaCa-2 cells. Autofluorescence images show that the FA-accumulated mitochondria also have abnormal NADH and FAD metabolism, likely induced by the dysfunction of the electron transport chain. We also found that when the cells were re-warmed to physiological temperature after a period of hypothermia, the FA-accumulated mitochondria changed their structural features, likely caused by the mitophagy process. To the best of our knowledge, this is the first time that FA accumulation in mitochondria was observed in live cells. Our research also demonstrates that multimodal label-free chemical imaging is an attractive tool to discover abnormal functions of mitochondria at the single-organelle level and can be used to quantify the dynamic changes of this organelle under perturbative conditions.

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