scholarly journals Label free 3D analysis of organelles in living cells by refractive index shows pre-mitotic organelle spinning in mammalian stem cells

2018 ◽  
Author(s):  
Patrick A. Sandoz ◽  
Christopher Tremblay ◽  
Sebastien Equis ◽  
Sorin Pop ◽  
Lisa Pollaro ◽  
...  
Keyword(s):  
Refractive Index ◽  
Lipid Droplets ◽  
Living Cells ◽  
Live Cells ◽  
3D Analysis ◽  
Label Free ◽  
Long Period ◽  
Microscopy Method ◽  
First Time ◽  
Cellular Organelles

AbstractHolo-tomographic microscopy (HTM) is a label-free non-phototoxic microscopy method reporting the fine changes of a cell’s refractive indexes (RI) in 3D. By combining HTM with epifluorescence, we demonstrate that cellular organelles such as Lipid droplets and mitochondria show a specific RI signature that distinguishes them with high resolution and contrast. We further show that HTM allows to follow in unprecedented ways the dynamics of mitochondria, lipid droplets as well as that of endocytic structures in live cells over long period of time, which led us to observe to our knowledge for the first time a global organelle spinning occurring before mitosis.

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.

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 Multiplex coherent anti-Stokes Raman scattering microspectroscopy detection of lipid droplets in cancer cells expressing TrkB

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Tiffany Guerenne-Del Ben ◽  
Vincent Couderc ◽  
Ludovic Duponchel ◽  
Vincent Sol ◽  
Philippe Leproux ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Lipid Droplets ◽  
Live Cells ◽  
Label Free ◽  
Colorectal Cancer Cell Lines ◽  
Invasive Method ◽  
Dye Staining ◽  
Cytoplasmic Lipid Droplets ◽  
Anti Stokes ◽  
Cell Fixation

Abstract For many years, scientists have been looking for specific biomarkers associated with cancer cells for diagnosis purposes. These biomarkers mainly consist of proteins located at the cell surface (e.g. the TrkB receptor) whose activation is associated with specific metabolic modifications. Identification of these metabolic changes usually requires cell fixation and specific dye staining. MCARS microspectroscopy is a label-free, non-toxic, and minimally invasive method allowing to perform analyses of live cells and tissues. We used this method to follow the formation of lipid droplets in three colorectal cancer cell lines expressing TrkB. MCARS images of cells generated from signal integration of CH2 stretching modes allow to discriminate between lipid accumulation in the endoplasmic reticulum and the formation of cytoplasmic lipid droplets. We found that the number of the latter was related to the TrkB expression level. This result was confirmed thanks to the creation of a HEK cell line which over-expresses TrkB. We demonstrated that BDNF-induced TrkB activation leads to the formation of cytoplasmic lipid droplets, which can be abolished by K252a, an inhibitor of TrkB. So, MCARS microspectroscopy proved useful in characterizing cancer cells displaying an aberrant lipid metabolism.

Pyrene appended thymine derivative for selective turn-on fluorescence sensing of uric acid in live cells

RSC Advances ◽  
2016 ◽  
Vol 6 (71) ◽  
pp. 66774-66778 ◽  
Author(s):  
Prithidipa Sahoo ◽  
Himadri Sekhar Sarkar ◽  
Sujoy Das ◽  
Kalipada Maiti ◽  
Md Raihan Uddin ◽  
...  
Keyword(s):  
Uric Acid ◽  
Fluorescent Probe ◽  
High Sensitivity ◽  
Living Cells ◽  
Live Cells ◽  
Fluorescence Sensing ◽  
Turn On ◽  
Sensitivity And Selectivity ◽  
First Time

A new ‘turn-ON’ fluorescent probe, pyrene appended thymine acetamide (PTA), with high sensitivity and selectivity for the detection of uric acid (UA) was developed and first time imaging of uric acid in living cells in water was achieved.

scholarly journals A Review of Refractometric Sensors Based on Long Period Fibre Gratings

2013 ◽  
Vol 2013 ◽  
pp. 1-14 ◽  
Author(s):  
G. Rego
Keyword(s):  
Thin Films ◽  
Refractive Index ◽  
Transition Region ◽  
Food Quality ◽  
Scientific Community ◽  
Surrounding Medium ◽  
Quality And Safety ◽  
Label Free ◽  
Long Period ◽  
Food Quality And Safety

In the last decade refractometric sensors have attracted an increasing interest by the scientific community due to their ability to perform ambient monitoring, to assess food quality and safety, and also to the fact that they enable the development of label free sensors in the biomedical area. These advances result, namely, from the use of long period fibre gratings in the turning points and/or with thin films in the transition region that allows resolutions of 10−6to changes in the refractive index of the surrounding medium. Resolutions exceeding 10−8can also be achieved when long period fibre gratings are combined with evanescent field based devices. This paper reviews the recent path towards the development of ultrahigh sensitive optical fibre refractometric sensors.

A dual-site two-photon fluorescent probe for visualizing mitochondrial aminothiols in living cells and mouse liver tissues

2016 ◽  
Vol 40 (9) ◽  
pp. 7399-7406 ◽  
Author(s):  
Fangfang Meng ◽  
Yong Liu ◽  
Xiaoqiang Yu ◽  
Weiying Lin
Keyword(s):  
Fluorescent Probe ◽  
Mouse Liver ◽  
Living Cells ◽  
Living Systems ◽  
Two Photon ◽  
Long Period ◽  
First Time ◽  
Dual Site ◽  
Liver Tissues

In this work, we developed a dual-site two-photon (TP) fluorescent RSH probe (CA-TPP) for imaging mitochondrial RSH in living systems. In particular, probe CA-TPP was capable of using TP fluorescence to track mitochondrial RSH over a long period for the first time.

scholarly journals Label-free digital holo-tomographic microscopy reveals virus-induced cytopathic effects in live cells

2018 ◽  
Author(s):  
Artur Yakimovich ◽  
Robert Witte ◽  
Vardan Andriasyan ◽  
Fanny Georgi ◽  
Urs F. Grebera
Keyword(s):  
Refractive Index ◽  
Membrane Dynamics ◽  
Live Cells ◽  
Refractive Index Gradient ◽  
Label Free ◽  
Virus Infections ◽  
Cytopathic Effects ◽  
Light Passing ◽  
Fluorescence Light ◽  
Simplex Virus

Cytopathic effects (CPEs) are a hallmark of infections. CPEs can be observed by phase contrast or fluorescence light microscopy, albeit at the cost of phototoxicity. We report that digital holo-tomographic microscopy (DHTM) reveals distinct patterns of virus infections in live cells with minimal perturbation. DHTM is label-free, and records the phase shift of low energy light passing through the specimen on a transparent surface. DHTM infers a 3-dimensional (3D) tomogram based on the refractive index (RI). By measuring RI and computing the refractive index gradient (RIG) values DHTM unveils on optical heterogeneity in cells upon virus infection. We find that vaccinia virus (VACV), herpes simplex virus (HSV) and rhinovirus (RV) infections progressively and distinctly increased RIG. VACV, but not HSV and RV infection induced oscillations of cell volume, while all three viruses altered cytoplasmic membrane dynamics, and induced apoptotic features akin to the chemical compound staurosporin, but with virus-specific signatures. In sum, we introduce DHTM for quantitative label-free microscopy in infection research, and uncover virus-type specific changes and CPE in living cells at minimal interference.

scholarly journals Tracking the formation and degradation of fatty-acid-accumulated mitochondria using label-free chemical imaging

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Chi Zhang ◽  
Stephen A. Boppart
Keyword(s):  
Fatty Acid ◽  
Structural Features ◽  
Chemical Imaging ◽  
Live Cells ◽  
Label Free ◽  
Regulatory Processes ◽  
Transport Chain ◽  
Abnormal Metabolism ◽  
Anti Stokes ◽  
First Time

AbstractThe mitochondrion is one of the key organelles for maintaining cellular homeostasis. External environmental stimuli and internal regulatory processes may 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 fluorescence to investigate 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 metabolism of nicotinamide adenine dinucleotide hydrogen, 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. 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 these organelles under perturbative conditions.

scholarly journals Dynamic signatures of lipid droplets as new markers to quantify cellular metabolic changes

2020 ◽  
Author(s):  
Chi Zhang ◽  
Stephen A. Boppart
Keyword(s):  
Lipid Droplets ◽  
Distribution Functions ◽  
Fluorescent Labeling ◽  
Metabolic Changes ◽  
Live Cells ◽  
Label Free ◽  
Metabolic States ◽  
Metabolic Properties ◽  
Displacement Speed ◽  
Anti Stokes

AbstractThe metabolic properties of live cells are very susceptible to intra- or extra-cellular perturbations, making their measurements challenging tasks. We show that the dynamics of lipid droplets (LDs) carry information to measure the lipid metabolism of live cells. Coherent anti-Stokes Raman scattering microscopy was used to statistically quantify LD dynamics in living cells in a label-free manner. We introduce dynamic signatures of cells derived from the LD displacement, speed, travel length, and directionality, which allows for the detection of cellular changes induced by stimuli such as fluorescent labeling, temperature change, starvation, and chemical treatment. Histogram fittings of the dynamic signatures using lognormal distribution functions provide quantification of changes in cellular metabolic states. The LD dynamics also enable separation of subpopulations of LDs correlated with different functions. We demonstrate that LD dynamics are new markers to quantify the metabolic changes in live cells.

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