scholarly journals Live Cell Partial Wave Spectroscopic microscopy: Label-free Imaging of the Native, Living Cellular Nano-architecture

2016 ◽  
Author(s):  
L. M. Almassalha ◽  
G. M. Bauer ◽  
J. Chandler ◽  
S. Gladstein ◽  
L. Cherkezya ◽  
...  
Keyword(s):  
Partial Wave ◽  
Chromatin Structure ◽  
Imaging Technique ◽  
Quantitative Imaging ◽  
Higher Order ◽  
Live Cell ◽  
Live Cells ◽  
Molecular Function ◽  
Metabolic Function ◽  
Label Free

AbstractThe organization of chromatin is a regulator of molecular processes including transcription, replication, and DNA repair. The structures within chromatin that regulate these processes span from the nucleosomal (10nm) to the chromosomal (>200nm) levels, with little known about the dynamics of chromatin structure between these scales due to a lack of quantitative imaging technique in live cells. Previous work using Partial Wave Spectroscopic (PWS) microscopy, a quantitative imaging technique with sensitivity to macromolecular organization between 20-200nm, has shown that transformation of chromatin at these length scales is a fundamental event during carcinogenesis. As the dynamics of chromatin likely play a critical regulatory role in cellular function, it is critical to develop live-cell imaging techniques that can probe the real-time temporal behavior of the chromatin nano-architecture. Therefore, we developed a live cell PWS technique which allows high-throughput, label-free study of the causal relationship between nanoscale organization and molecular function in real-time. In this work, we employ live cell PWS to study the change in chromatin structure due to DNA damage and expand on the link between metabolic function and the structure of higher-order chromatin. In particular, we studied the temporal changes to chromatin during UV light exposure, show that live cell DNA binding dyes induce damage to chromatin within seconds, and demonstrate a direct link between higher-order chromatin structure and mitochondrial membrane potential. Since biological function is tightly paired with structure, live cell PWS is a powerful tool to study the nanoscale structure-function relationship in live cells.Significance StatementChromatin is one of the most critical structures within the cell because it houses most genetic information. Its structure is well understood at the nucleosomal (<20nm) and chromosomal (>200nm) levels, however, due to the lack of quantitative imaging modalities to study this organization, little is known about the higher-order structure between these length scales in live cells. We present a label-free technique, live cell Partial Wave Spectroscopic (PWS) microscopy with sensitivity to structures between 20-200nm that can quantify the nano-architecture in live cells. With this technique, we can detect DNA fragmentation and expand on the link between metabolic function and higher-order chromatin structure. Live cell PWS allows high-throughput, label-free study of the causal relationship between nanoscale organization and molecular function in live cells.

scholarly journals Label-free imaging of the native, living cellular nanoarchitecture using partial-wave spectroscopic microscopy

2016 ◽  
Vol 113 (42) ◽  
pp. E6372-E6381 ◽  
Author(s):  
Luay M. Almassalha ◽  
Greta M. Bauer ◽  
John E. Chandler ◽  
Scott Gladstein ◽  
Lusik Cherkezyan ◽  
...  
Keyword(s):  
Real Time ◽  
Partial Wave ◽  
Chromatin Structure ◽  
Imaging Technique ◽  
Light Exposure ◽  
Quantitative Imaging ◽  
Higher Order ◽  
Live Cell ◽  
Live Cells ◽  
Label Free

The organization of chromatin is a regulator of molecular processes including transcription, replication, and DNA repair. The structures within chromatin that regulate these processes span from the nucleosomal (10-nm) to the chromosomal (>200-nm) levels, with little known about the dynamics of chromatin structure between these scales due to a lack of quantitative imaging technique in live cells. Previous work using partial-wave spectroscopic (PWS) microscopy, a quantitative imaging technique with sensitivity to macromolecular organization between 20 and 200 nm, has shown that transformation of chromatin at these length scales is a fundamental event during carcinogenesis. As the dynamics of chromatin likely play a critical regulatory role in cellular function, it is critical to develop live-cell imaging techniques that can probe the real-time temporal behavior of the chromatin nanoarchitecture. Therefore, we developed a live-cell PWS technique that allows high-throughput, label-free study of the causal relationship between nanoscale organization and molecular function in real time. In this work, we use live-cell PWS to study the change in chromatin structure due to DNA damage and expand on the link between metabolic function and the structure of higher-order chromatin. In particular, we studied the temporal changes to chromatin during UV light exposure, show that live-cell DNA-binding dyes induce damage to chromatin within seconds, and demonstrate a direct link between higher-order chromatin structure and mitochondrial membrane potential. Because biological function is tightly paired with structure, live-cell PWS is a powerful tool to study the nanoscale structure–function relationship in live cells.

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.

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 and live cell imaging by interferometric scattering microscopy

2018 ◽  
Vol 9 (10) ◽  
pp. 2690-2697 ◽  
Author(s):  
Jin-Sung Park ◽  
Il-Buem Lee ◽  
Hyeon-Min Moon ◽  
Jong-Hyeon Joo ◽  
Kyoung-Hoon Kim ◽  
...  
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Complex Structure ◽  
Live Cell ◽  
Fluorescent Label ◽  
Live Cells ◽  
Label Free ◽  
Cytoplasmic Organelles ◽  
Microscopic Techniques

Despite recent remarkable advances in microscopic techniques, it still remains very challenging to directly observe the complex structure of cytoplasmic organelles in live cells without a fluorescent label.

scholarly journals Revealing architectural order with quantitative label-free imaging and deep learning

2019 ◽  
Author(s):  
Syuan-Ming Guo ◽  
Li-Hao Yeh ◽  
Jenny Folkesson ◽  
Ivan Ivanov ◽  
Anitha Priya Krishnan ◽  
...  
Keyword(s):  
Quantitative Imaging ◽  
Three Dimensional ◽  
Kidney Tissue ◽  
Live Cells ◽  
Label Free ◽  
Computationally Efficient ◽  
Human Brain Tissue ◽  
Tissue Slices ◽  
Tissue Sections ◽  
Scalable Analysis

Quantitative imaging of biological architecture with fluorescent labels is not as scalable as genomic or proteomic measurements. Here, we combine quantitative label-free imaging and deep neural networks for scalable analysis of complex structures. We reconstruct quantitative three-dimensional density, anisotropy, and orientation in live cells and tissue slices from polarization- and depth-resolved images. We report a computationally efficient variant of U-Net architecture that predicts a 3D fluorescent structure from its morphology and physical properties. We evaluate the performance of our models by predicting F-actin and nuclei in mouse kidney tissue. Further, we report label-free imaging of axon tracts and predict level of myelination in human brain tissue sections. We demonstrate the model's ability to rescue inconsistent labeling. We anticipate that the proposed approach will enable quantitative analysis of architectural order across scales of organelles to tissues.

scholarly journals Label-free high-resolution 3-D imaging of gold nanoparticles inside live cells using optical diffraction tomography

2016 ◽  
Author(s):  
Doyeon Kim ◽  
Nuri Oh ◽  
Kyoohyun Kim ◽  
SangYun Lee ◽  
Chan-Gi Pack ◽  
...  
Keyword(s):  
Gold Nanoparticles ◽  
Refractive Index ◽  
Quantitative Imaging ◽  
Extended Period ◽  
Optical Diffraction ◽  
Live Cells ◽  
Diffraction Tomography ◽  
Label Free ◽  
Optical Diffraction Tomography ◽  
4T1 Cells

AbstractDelivery of gold nanoparticles (GNPs) into live cells has high potentials, ranging from molecular-specific imaging, photodiagnostics, to photothermal therapy. However, studying the long-term dynamics of cells with GNPs using conventional fluorescence techniques suffers from phototoxicity and photobleaching. Here, we present a method for 3-D imaging of GNPs inside live cells exploiting refractive index (RI) as imaging contrast. Employing optical diffraction tomography, 3-D RI tomograms of live cells with GNPs are precisely measured for an extended period with sub-micrometer resolution. The locations and contents of GNPs in live cells are precisely addressed and quantified due to their distinctly high RI values, which was validated by confocal fluorescence imaging of fluorescent dye conjugated GNPs. In addition, we perform quantitative imaging analysis including the segmentations of GNPs in the cytosol, the volume distributions of aggregated GNPs, and the temporal evolution of GNPs contents in HeLa and 4T1 cells.AbbreviationsGNPsgold nanoparticlesRIrefractive indexODToptical diffraction tomographyDMDdigital micromirror device

scholarly journals Dissecting lipid droplet biology with coherent Raman scattering microscopy

2021 ◽  
Vol 135 (5) ◽  
Author(s):  
Tao Chen ◽  
Ahmet Yavuz ◽  
Meng C. Wang
Keyword(s):  
Raman Scattering ◽  
Lipid Droplets ◽  
Quantitative Imaging ◽  
Live Cells ◽  
Label Free ◽  
Genetic Screens ◽  
Spatiotemporal Resolution ◽  
Coherent Raman Scattering ◽  
Coherent Raman

ABSTRACT Lipid droplets (LDs) are lipid-rich organelles universally found in most cells. They serve as a key energy reservoir, actively participate in signal transduction and dynamically communicate with other organelles. LD dysfunction has been associated with a variety of diseases. The content level, composition and mobility of LDs are crucial for their physiological and pathological functions, and these different parameters of LDs are subject to regulation by genetic factors and environmental inputs. Coherent Raman scattering (CRS) microscopy utilizes optical nonlinear processes to probe the intrinsic chemical bond vibration, offering label-free, quantitative imaging of lipids in vivo with high chemical specificity and spatiotemporal resolution. In this Review, we provide an overview over the principle of CRS microscopy and its application in tracking different parameters of LDs in live cells and organisms. We also discuss the use of CRS microscopy in genetic screens to discover lipid regulatory mechanisms and in understanding disease-related lipid pathology.

scholarly journals Ptychography – a label free, high-contrast imaging technique for live cells using quantitative phase information

2013 ◽  
Vol 3 (1) ◽  
Author(s):  
Joanne Marrison ◽  
Lotta Räty ◽  
Poppy Marriott ◽  
Peter O'Toole
Keyword(s):  
Imaging Technique ◽  
Live Cells ◽  
High Contrast ◽  
Label Free ◽  
Phase Information ◽  
Contrast Imaging ◽  
Quantitative Phase ◽  
High Contrast Imaging

α-Methylene-β-Lactone Probe for Measuring Live-Cell Reactions of Small Molecules

2020 ◽  
Author(s):  
Lei Wang ◽  
Louis Riel ◽  
Bekim Bajrami ◽  
Bin Deng ◽  
Amy Howell ◽  
...  
Keyword(s):  
Mass Spectra ◽  
Live Cell ◽  
Live Cells ◽  
The Novel ◽  
Proteomics Analysis ◽  
Chemical Proteomics ◽  
Tandem Mass Spectra ◽  
Modified Peptides ◽  
Covalent Probes ◽  
Ht 29

The novel use of the α-methylene-β-lactone (MeLac) moiety as a warhead of multiple electrophilic sites is reported. In this study, we demonstrate that a MeLac-alkyne is a competent covalent probe and reacts with diverse proteins in live cells. Proteomics analysis of affinity-enriched samples identifies probe-reacted proteins, resolves their modified peptides/residues, and thus characterizes probe-protein reactions. Unique methods are developed to evaluate confidence in the identification of the reacted proteins and modified peptides. Tandem mass spectra of the peptides reveal that MeLac reacts with nucleophilic cysteine, serine, lysine, threonine, and tyrosine residues, through either Michael addition or acyl addition. A peptide-centric proteomics platform, using MeLac-alkyne as the measurement probe, successfully analyzes the Orlistat selectivity in live HT-29 cells. MeLac is a versatile warhead demonstrating enormous potential to expedite the development of covalent probes and inhibitors in interrogating protein (re)activity. MeLac-empowered platforms in chemical proteomics are widely adaptable for measuring the live-cell action of reactive molecules.

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