scholarly journals Phase Measurement Using DIC Microscopy

2017 ◽  
Vol 23 (2) ◽  
pp. 629-643
Author(s):  
Krisztian Koos ◽  
Begüm Peksel ◽  
Lóránd Kelemen
Keyword(s):  
Phase Measurement ◽  
Surrounding Medium ◽  
Optical Path Length ◽  
Computational Method ◽  
Live Cells ◽  
Label Free ◽  
Transparent Objects ◽  
Morphological Differences ◽  
Microscopy Techniques ◽  
Dic Microscopy

The development of fluorescent probes and proteins has helped make light microscopy more popular by allowing the visualization of specific subcellular components, location and dynamics of biomolecules. However, it is not always feasible to label the cells as it may be phototoxic or perturb their functionalities. Label-free microscopy techniques allow us to work with live cells without perturbation and to evaluate morphological differences, which in turn can provide useful information for high-throughput assays. In this study, we use one of the most popular label-free techniques called differential interference contrast (DIC) microscopy to estimate the phase of cells and other nearly transparent objects and instantly estimate their height. DIC images provide detailed information about the optical path length (OPL) differences in the sample and they are visually similar to a gradient image. Our previous DIC construction algorithm outputs an image where the values are proportional to the OPL (or implicitly the phase) of the sample. Although the reconstructed images are capable of describing cellular morphology and to a certain extent turn DIC into a quantitative technique, the actual OPL has to be computed from the input DIC image and the microscope calibration settings. Here we propose a computational method to measure the phase and approximate height of cells after microscope calibration, assuming a linear formation model. After a calibration step the phase of further samples can be determined when the refractive indices of the sample and the surrounding medium is known. The precision of the method is demonstrated on reconstructing the thickness of known objects and real cellular samples.

scholarly journals Utilizing Stimulated Raman Scattering Microscopy To Study Intracellular Distribution of Label-Free Ponatinib in Live Cells

2019 ◽  
Vol 63 (5) ◽  
pp. 2028-2034 ◽  
Author(s):  
Kristel Sepp ◽  
Martin Lee ◽  
Marie T. J. Bluntzer ◽  
G. Vignir Helgason ◽  
Alison N. Hulme ◽  
...  
Keyword(s):  
Raman Scattering ◽  
Stimulated Raman Scattering ◽  
Intracellular Distribution ◽  
Live Cells ◽  
Label Free ◽  
Stimulated Raman

scholarly journals Microfluidic Tools for Enhanced Characterization of Therapeutic Stem Cells and Prediction of Their Potential Antimicrobial Secretome

Antibiotics ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 750
Author(s):  
Pasquale Marrazzo ◽  
Valeria Pizzuti ◽  
Silvia Zia ◽  
Azzurra Sargenti ◽  
Daniele Gazzola ◽  
...  
Keyword(s):  
Stem Cells ◽  
Defense Mechanisms ◽  
Live Cells ◽  
Label Free ◽  
Bacterial Diseases ◽  
Prospective Application ◽  
Therapy Strategies ◽  
Stromal Stem Cells ◽  
Different Sources

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.

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

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.

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

mSphere ◽  
2018 ◽  
Vol 3 (6) ◽  
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.

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

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.

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

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.

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

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.

scholarly journals Mitotic Chromosomes in Live Cells Characterized Using High-Speed and Label-Free Optical Diffraction Tomography

Cells ◽  
2019 ◽  
Vol 8 (11) ◽  
pp. 1368 ◽  
Author(s):  
Kim ◽  
Lee ◽  
Fujii ◽  
Lee ◽  
Lee ◽  
...  
Keyword(s):  
Diffusion Coefficient ◽  
Three Dimensional ◽  
Laser Scanning Microscopy ◽  
Optical Diffraction ◽  
Live Cells ◽  
Confocal Laser ◽  
Diffraction Tomography ◽  
Label Free ◽  
Mitotic Chromosomes ◽  
Optical Diffraction Tomography

The cell nucleus is a three-dimensional, dynamic organelle organized into subnuclear compartments such as chromatin and nucleoli. The structure and function of these compartments are maintained by diffusion and interactions between related factors as well as by dynamic and structural changes. Recent studies using fluorescent microscopic techniques suggest that protein factors can access and are freely mobile in heterochromatin and in mitotic chromosomes, despite their densely packed structure. However, the physicochemical properties of the chromosome during cell division are not fully understood. In the present study, characteristic properties such as the refractive index (RI), volume of the mitotic chromosomes, and diffusion coefficient (D) of fluorescent probes inside the chromosome were quantified using an approach combining label-free optical diffraction tomography with complementary confocal laser-scanning microscopy and fluorescence correlation spectroscopy. Variations in these parameters correlated with osmotic conditions, suggesting that changes in RI are consistent with those of the diffusion coefficient for mitotic chromosomes and cytosol. Serial RI tomography images of chromosomes in live cells during mitosis were compared with three-dimensional confocal micrographs to demonstrate that compaction and decompaction of chromosomes induced by osmotic change were characterized by linked changes in chromosome RI, volume, and the mobilities of fluorescent proteins.

Sign in / Sign up

Export Citation Format

Share Document