Jijo Lukose, Mithun N, Ganesh Mohan, Shamee Shastry and Santhosh Chidangil
The use of normal saline for washing red blood cells and treating critically ill patients is a regular medical practice in hospital settings. An optical tweezer in combination with Raman spectroscopy is an analytical tool employed for the investigation of single cell dynamics, thus providing molecular fingerprint of the cell by optically trapping the cell at a laser focus. In this study, the impact of normal saline on individual human red blood cell was compared with that of blood plasma using Raman tweezers spectroscopy. Major spectral variations in the marker frequencies at 1209 cm−1, 1222 cm−1, 1544 cm−1, and 1561 cm−1 of the Raman spectrum of the treated cells imply that the transition of hemoglobin to the deoxygenated state occurs when 0.9% normal saline is used. This may result in serious implications in blood transfusion. The results obtained from the principal component analysis also displayed clear differentiation among the red blood cells diluted in normal saline and those diluted in plasma. In future studies, efforts will be made to correlate the deoxygenation status of red blood cells with various human disorders.
DOI
Tuesday, April 2, 2019
High-speed transverse and axial optical force measurements using amplitude filter masks
Anatolii V. Kashchuk, Timo A. Nieminen, Halina Rubinsztein-Dunlop, and Alexander B. Stilgoe
Direct optical force measurement is a versatile method used in optical tweezers experiments, providing accurate measurements of forces for a wide range of particles and trapping beams. It is based on the detection of the change of the momentum of light scattered by a trapped object. A digital micromirror device can be used to selectively reflect light in different directions using an appropriately defined mask. We have developed position-sensitive masked detection (PSMD) for measuring transverse (radial) and axial forces. The method is comparable in performance to the fastest split detectors, while maintaining the linearity and customizability similar to duo-lateral position-sensitive detectors (PSD) and cameras. We show an order of magnitude increase in the bandwidth compared to a conventional PSD for radial forces. We measure axial force and verify the measurement using the Stokes drag for the particle. Combining both detectors (PSMD and PSD), we can perform full 3-D optical force measurements in real time.
DOI
Direct optical force measurement is a versatile method used in optical tweezers experiments, providing accurate measurements of forces for a wide range of particles and trapping beams. It is based on the detection of the change of the momentum of light scattered by a trapped object. A digital micromirror device can be used to selectively reflect light in different directions using an appropriately defined mask. We have developed position-sensitive masked detection (PSMD) for measuring transverse (radial) and axial forces. The method is comparable in performance to the fastest split detectors, while maintaining the linearity and customizability similar to duo-lateral position-sensitive detectors (PSD) and cameras. We show an order of magnitude increase in the bandwidth compared to a conventional PSD for radial forces. We measure axial force and verify the measurement using the Stokes drag for the particle. Combining both detectors (PSMD and PSD), we can perform full 3-D optical force measurements in real time.
DOI
Rab6 regulates cell migration and invasion by recruiting Cdc42 and modulating its activity
Katharina Vestre, Ingrid Kjos, Noemi Antonella Guadagno, Marita Borg Distefano, Felix Kohler, Federico Fenaroli, Oddmund Bakke, Cinzia Progida
Rab proteins are master regulators of intracellular membrane trafficking, but they also contribute to cell division, signaling, polarization, and migration. The majority of the works describing the mechanisms used by Rab proteins to regulate cell motility involve intracellular transport of key molecules important for migration. Interestingly, a few studies indicate that Rabs can modulate the activity of Rho GTPases, important regulators for the cytoskeleton rearrangements, but the mechanisms behind this crosstalk are still poorly understood. In this work, we identify Rab6 as a negative regulator of cell migration in vitro and in vivo. We show that the loss of Rab6 promotes formation of actin protrusions and influences actomyosin dynamics by upregulating Cdc42 activity and downregulating myosin II phosphorylation. We further provide the molecular mechanism behind this regulation demonstrating that Rab6 interacts with both Cdc42 and Trio, a GEF for Cdc42. In sum, our results uncover a mechanism used by Rab proteins to ensure spatial regulation of Rho GTPase activity for coordination of cytoskeleton rearrangements required in migrating cells.
Rab proteins are master regulators of intracellular membrane trafficking, but they also contribute to cell division, signaling, polarization, and migration. The majority of the works describing the mechanisms used by Rab proteins to regulate cell motility involve intracellular transport of key molecules important for migration. Interestingly, a few studies indicate that Rabs can modulate the activity of Rho GTPases, important regulators for the cytoskeleton rearrangements, but the mechanisms behind this crosstalk are still poorly understood. In this work, we identify Rab6 as a negative regulator of cell migration in vitro and in vivo. We show that the loss of Rab6 promotes formation of actin protrusions and influences actomyosin dynamics by upregulating Cdc42 activity and downregulating myosin II phosphorylation. We further provide the molecular mechanism behind this regulation demonstrating that Rab6 interacts with both Cdc42 and Trio, a GEF for Cdc42. In sum, our results uncover a mechanism used by Rab proteins to ensure spatial regulation of Rho GTPase activity for coordination of cytoskeleton rearrangements required in migrating cells.
Effects of caffeine on the structure and conformation of DNA: A force spectroscopy study
T. A. Moura, L. Oliveira, M. S. Rocha
Here, we use single molecule force spectroscopy performed with optical tweezers in order to investigate the interaction between Caffeine and the DNA molecule for various different concentrations of the alkaloid and under two distinct ionic strengths of the surrounding buffer. We were able to determine the mechanical changes induced on the double-helix structure due to Caffeine binding, the binding mode and the binding parameters of the interaction. The results obtained show that Caffeine binds to DNA by outside the double-helix with a higher affinity at lower ionic strengths. On the other hand, a considerable cooperativity was found only for sufficient high ionic strengths, suggesting that Caffeine may binding forming dimers and/or trimers along the double-helix under this condition. Finally, it was also shown that Caffeine stabilizes the DNA double-helix upon binding, preventing force-induced DNA melting.
DOI
Here, we use single molecule force spectroscopy performed with optical tweezers in order to investigate the interaction between Caffeine and the DNA molecule for various different concentrations of the alkaloid and under two distinct ionic strengths of the surrounding buffer. We were able to determine the mechanical changes induced on the double-helix structure due to Caffeine binding, the binding mode and the binding parameters of the interaction. The results obtained show that Caffeine binds to DNA by outside the double-helix with a higher affinity at lower ionic strengths. On the other hand, a considerable cooperativity was found only for sufficient high ionic strengths, suggesting that Caffeine may binding forming dimers and/or trimers along the double-helix under this condition. Finally, it was also shown that Caffeine stabilizes the DNA double-helix upon binding, preventing force-induced DNA melting.
DOI
Tunable optical assembly of subwavelength particles by a microfiber cavity
Yang Yu, Tinghui Xiao, Yuan-Xing Li, Qing-Guang Zeng, Bing-Qian Li and Zhi-Yuan Li
Optical assembly as a multiple optical trapping technique enables patterned arrangements of matters ranging from atoms to microparticles for diverse applications in biophysics, quantum physics, surface chemistry, and cell biology. Optical potential energy landscapes based on evanescent fields are conventionally employed for optical assembly of subwavelength particles, but are typically limited to predefined patterns and lacking in tunability. Here we present a microfiber photonic crystal cavity applicable for tunable optical assembly of subwavelength particles along a flexible path. This is enabled by excellent mechanical flexibility of the microfiber cavity as well as its broadband photonic crystal reflectors. By virtue of the broadband reflectors, the lattice constant of the assembled particles is precisely tunable via altering the wavelength of input light. Three-dimensional optical assembly is also realized by making use of the high-order transverse mode of the microfiber cavity. Moreover, the optical assembly process is detectable by simply monitoring the reflection/transmission spectrum of the microfiber cavity. The design of the microfiber cavity heralds a new way for tunable optical assembly of subwavelength particles, potentially applicable for development of tunable photonic crystals, metamaterials, and sensors.
Nanowire-type plasmonic waveguides as strong and tunable optical tweezers
Shu Yang and Kang Zhao
A series of nanowire-type plasmonic waveguides are proposed. The mode properties of these waveguides and their dependences on various geometry parameters are studied. It is shown that they can generate deep subwavelength confinement and long-range propagation simultaneously. Moreover, the optical forces exerted on dielectric nanoparticles by these waveguides are calculated. It is found that the optical trapping forces are very strong, and that their distribution can be effectively regulated by certain geometry parameters. Using these features, strong and tunable near-field optical tweezers can be designed.
DOI
A series of nanowire-type plasmonic waveguides are proposed. The mode properties of these waveguides and their dependences on various geometry parameters are studied. It is shown that they can generate deep subwavelength confinement and long-range propagation simultaneously. Moreover, the optical forces exerted on dielectric nanoparticles by these waveguides are calculated. It is found that the optical trapping forces are very strong, and that their distribution can be effectively regulated by certain geometry parameters. Using these features, strong and tunable near-field optical tweezers can be designed.
DOI
Monday, April 1, 2019
Trapping two types of Rayleigh particles using a focused partially coherent anomalous vortex beam
Miao Dong, Dagang Jiang, Nanhang Luo, Yuanjie Yang
The radiation forces of focused partially coherent anomalous vortex (AV) beams on Rayleigh particles of different refractive indices are studied theoretically and numerically. The influences of the topological charge, the beam order and coherence length on the radiation force are also discussed. It is shown that the focused partially coherent AV beam can be used to trap high index of refraction particles at the focus and to trap low index of refraction particles in the vicinity of the focus. It is also found that the radiation force can be modulated by the topological charge, the beam order and the coherence length.
DOI
The radiation forces of focused partially coherent anomalous vortex (AV) beams on Rayleigh particles of different refractive indices are studied theoretically and numerically. The influences of the topological charge, the beam order and coherence length on the radiation force are also discussed. It is shown that the focused partially coherent AV beam can be used to trap high index of refraction particles at the focus and to trap low index of refraction particles in the vicinity of the focus. It is also found that the radiation force can be modulated by the topological charge, the beam order and the coherence length.
DOI
Mechanical properties of RBCs under oxidative stress measured by optical tweezers
Jiaqi Liu, Fan Zhang, Lianqing Zhu, Daping Chu, Xinghua Qu
Mechanical characterization of human red blood cells (RBC) is crucial to the study of the pathology, drug screening, clinical diagnosis, and treatment of Parkinson’s disease. Oxidative stress has been found to be a dominant factor among the causes of Parkinson’s disease. This paper proposes a method for measuring cell mechanical properties using optical tweezers. By measuring the membrane shear modulus of RBCs treated with hydrogen peroxide (O2) at different concentrations, mechanical properties of RBCs under oxidative stress were analyzed. The experimental results reveal that increased membrane shear modulus and decreased cell deformability are commensurate with increasing concentrations of O2. This proposal will benefit both basic research and clinical applications in the field of Parkinson’s disease research. The results will also provide a guideline for measuring the mechanical properties of other cells.
DOI
Mechanical characterization of human red blood cells (RBC) is crucial to the study of the pathology, drug screening, clinical diagnosis, and treatment of Parkinson’s disease. Oxidative stress has been found to be a dominant factor among the causes of Parkinson’s disease. This paper proposes a method for measuring cell mechanical properties using optical tweezers. By measuring the membrane shear modulus of RBCs treated with hydrogen peroxide (O2) at different concentrations, mechanical properties of RBCs under oxidative stress were analyzed. The experimental results reveal that increased membrane shear modulus and decreased cell deformability are commensurate with increasing concentrations of O2. This proposal will benefit both basic research and clinical applications in the field of Parkinson’s disease research. The results will also provide a guideline for measuring the mechanical properties of other cells.
DOI
Molecular Crowding Tunes Material States of Ribonucleoprotein Condensates
Taranpreet Kaur, Ibraheem Alshareedah, Wei Wang, Jason Ngo, Mahdi Muhammad Moosa and Priya R. Banerjee
Ribonucleoprotein (RNP) granules are membraneless liquid condensates that dynamically form, dissolve, and mature into a gel-like state in response to a changing cellular environment. RNP condensation is largely governed by promiscuous attractive inter-chain interactions mediated by low-complexity domains (LCDs). Using an archetypal disordered RNP, fused in sarcoma (FUS), here we study how molecular crowding impacts the RNP liquid condensation. We observe that the liquid–liquid coexistence boundary of FUS is lowered by polymer crowders, consistent with an excluded volume model. With increasing bulk crowder concentration, the RNP partition increases and the diffusion rate decreases in the condensed phase. Furthermore, we show that RNP condensates undergo substantial hardening wherein protein-dense droplets transition from viscous fluid to viscoelastic gel-like states in a crowder concentration-dependent manner. Utilizing two distinct LCDs that broadly represent commonly occurring sequence motifs driving RNP phase transitions, we reveal that the impact of crowding is largely independent of LCD charge and sequence patterns. These results are consistent with a thermodynamic model of crowder-mediated depletion interaction, which suggests that inter-RNP attraction is enhanced by molecular crowding. The depletion force is likely to play a key role in tuning the physical properties of RNP condensates within the crowded cellular space.
Ribonucleoprotein (RNP) granules are membraneless liquid condensates that dynamically form, dissolve, and mature into a gel-like state in response to a changing cellular environment. RNP condensation is largely governed by promiscuous attractive inter-chain interactions mediated by low-complexity domains (LCDs). Using an archetypal disordered RNP, fused in sarcoma (FUS), here we study how molecular crowding impacts the RNP liquid condensation. We observe that the liquid–liquid coexistence boundary of FUS is lowered by polymer crowders, consistent with an excluded volume model. With increasing bulk crowder concentration, the RNP partition increases and the diffusion rate decreases in the condensed phase. Furthermore, we show that RNP condensates undergo substantial hardening wherein protein-dense droplets transition from viscous fluid to viscoelastic gel-like states in a crowder concentration-dependent manner. Utilizing two distinct LCDs that broadly represent commonly occurring sequence motifs driving RNP phase transitions, we reveal that the impact of crowding is largely independent of LCD charge and sequence patterns. These results are consistent with a thermodynamic model of crowder-mediated depletion interaction, which suggests that inter-RNP attraction is enhanced by molecular crowding. The depletion force is likely to play a key role in tuning the physical properties of RNP condensates within the crowded cellular space.
Mucus Microrheology Measured on Human Bronchial Epithelium Culture
Myriam Jory, Karim Bellouma, Christophe Blanc, Laura Casanellas, Aurélie Petit, Paul Reynaud, Charlotte Vernisse, Isabelle Vachier, Arnaud Bourdin and Gladys Massiera
We describe an original method to measure mucus microrheology on human bronchial epithelium culture using optical tweezers. We probed rheology on the whole thickness of mucus above the epithelium and showed that mucus gradually varies in rheological response, from an elastic behavior close to the epithelium to a viscous one far away. Microrheology was also performed on mucus collected on the culture, on ex vivo mucus collected by bronchoscopy, and on another epithelium model. Differences are discussed and are related to mucus heterogeneity, adhesiveness, and collection method.
DOI
We describe an original method to measure mucus microrheology on human bronchial epithelium culture using optical tweezers. We probed rheology on the whole thickness of mucus above the epithelium and showed that mucus gradually varies in rheological response, from an elastic behavior close to the epithelium to a viscous one far away. Microrheology was also performed on mucus collected on the culture, on ex vivo mucus collected by bronchoscopy, and on another epithelium model. Differences are discussed and are related to mucus heterogeneity, adhesiveness, and collection method.
DOI
Structural and functional studies of erythrocyte membrane-skeleton by single-cell and single-molecule techniques
Fulin Xing, Fen Hu, Jianyu Yang, Leiting Pan and Jingjun Xu
As the indispensable oxygen-transporting cells, erythrocytes exhibit extreme deformability and amazing stability as they are subject to huge reversible shear stress and extrusion force during massive circulation in the body. The unique architecture of spectrin-actin-based membrane-skeleton is considered to be responsible for such excellent mechanical properties of erythrocytes. Although erythrocytes have been recognized for more than 300 years, myriad questions about membrane-skeleton constantly attract people’s attention. Here, we summarize the kinds of distinctive single-cell and single-molecule techniques that were used to investigate the structure and function of erythrocyte membrane-skeleton at macro and micro levels.
As the indispensable oxygen-transporting cells, erythrocytes exhibit extreme deformability and amazing stability as they are subject to huge reversible shear stress and extrusion force during massive circulation in the body. The unique architecture of spectrin-actin-based membrane-skeleton is considered to be responsible for such excellent mechanical properties of erythrocytes. Although erythrocytes have been recognized for more than 300 years, myriad questions about membrane-skeleton constantly attract people’s attention. Here, we summarize the kinds of distinctive single-cell and single-molecule techniques that were used to investigate the structure and function of erythrocyte membrane-skeleton at macro and micro levels.
Label‐free differentiation of human immunodeficiency virus‐1 infected from uninfected cells using transmission measurement
Saturnin Ombinda‐Lemboumba, Rudzani Malabi, Masixole Y. Lugongolo, Lebogang Thobakgale, Sello L. Manoto, Patience Mthunzi‐Kufa
Transmission measurement has been perceived as a potential candidate for label‐free investigation of biological material. It is a real‐time, label‐free and non‐invasive optical detection technique that has found wide applications in pharmaceutical industry as well as the biological and medical fields. Combining transmission measurement with optical trapping has emerged as a powerful tool allowing stable sample trapping, while also facilitating transmittance data analysis. In this study, a near‐infrared laser beam emitting at a wavelength of 1064 nm was used for both optical trapping and transmission measurement investigation of human immunodeficiency virus 1 (HIV‐1) infected and uninfected TZM‐bl cells. The measurements of the transmittance intensity of individual cells in solution were carried out using a home built optical trapping system combined with laser transmission setup using a single beam gradient trap. Transmittance spectral intensity patterns revealed significant differences between the HIV‐1 infected and uninfected cells. This result suggests that the transmittance data analysis technique used in this study has the potential to differentiate between infected and uninfected TZM‐bl cells without the use of labels. The results obtained in this study could pave a way into developing an HIV‐1 label‐free diagnostic tool with possible applications at the point of care
DOI
Transmission measurement has been perceived as a potential candidate for label‐free investigation of biological material. It is a real‐time, label‐free and non‐invasive optical detection technique that has found wide applications in pharmaceutical industry as well as the biological and medical fields. Combining transmission measurement with optical trapping has emerged as a powerful tool allowing stable sample trapping, while also facilitating transmittance data analysis. In this study, a near‐infrared laser beam emitting at a wavelength of 1064 nm was used for both optical trapping and transmission measurement investigation of human immunodeficiency virus 1 (HIV‐1) infected and uninfected TZM‐bl cells. The measurements of the transmittance intensity of individual cells in solution were carried out using a home built optical trapping system combined with laser transmission setup using a single beam gradient trap. Transmittance spectral intensity patterns revealed significant differences between the HIV‐1 infected and uninfected cells. This result suggests that the transmittance data analysis technique used in this study has the potential to differentiate between infected and uninfected TZM‐bl cells without the use of labels. The results obtained in this study could pave a way into developing an HIV‐1 label‐free diagnostic tool with possible applications at the point of care
DOI
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