Biomechanics of Ex Vivo-Generated Red Blood Cells Investigated by Optical Tweezers and Digital Holographic Microscopy

Bernecker, Claudia; Lima, Maria; Ciubotaru, Catalin D.; Schlenke, Peter; Dorn, Isabel; Cojoc, Dan

doi:10.3390/cells10030552

Cited by 15 publications

(16 citation statements)

References 52 publications

(61 reference statements)

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“…After placement on the cell body, large moving EVs could be recaptured by the laser trap (Table 1, n = 10/10), proving that they were outside neurons, whereas only 27% of ‘static’ EVs, that remained adherent to the place of delivery, were recaptured by optical tweezers, being transiently attracted towards the laser (Table 1, n = 4/15). Although we could not exclude that the laser trapping force (maximum 50 piconewton for our setup) might not be able to re‐trap EVs strongly adhering to the neuron surface (Ashkin, 1992; Bernecker et al., 2021), this suggested that a fraction of large EVs may undergo internalization into the neuron cell body. To assess this hypothesis, we labelled large EVs with the fluorescent dye mCLING (Brenna et al., 2020) and analysed by confocal microscopy the localization of mCLING‐labelled large EVs 1 h after in bulk addition to neurons transfected with membrane‐targeted GFP.…”

Section: Resultsmentioning

confidence: 97%

Astrocytes‐derived extracellular vesicles in motion at the neuron surface: Involvement of the prion protein

D’Arrigo

Gabrielli

Scaroni

et al. 2021

J of Extracellular Vesicle

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Astrocytes-derived extracellular vesicles (EVs) are key players in glia-neuron communication. However, whether EVs interact with neurons at preferential sites and how EVs reach these sites on neurons remains elusive. Using optical manipulation to study single EV-neuron dynamics, we here show that large EVs scan the neuron surface and use neuronal processes as highways to move extracellularly. Large EV motion on neurites is driven by the binding of EV to a surface receptor that slides on neuronal membrane, thanks to actin cytoskeleton rearrangements. The use of prion protein (PrP)-coated synthetic beads and PrP knock out EVs/neurons points at vesicular PrP and its receptor(s) on neurons in the control of EV motion. Surprisingly, a fraction of large EVs contains actin filaments and has an independent capacity to move in an actin-mediated way, through intermittent contacts with the plasma membrane. Our results unveil, for the first time, a dual mechanism exploited by astrocytic large EVs to passively/actively reach target sites on neurons moving on the neuron surface. K E Y WO R D Sastrocytes, cytoskeleton, extracellular vesicles, neurons, optical tweezers, prion protein, PrP knock-out  INTRODUCTIONExtracellular vesicles (EVs) are circular membrane fragments released by all cells which function as intercellular signalling vehicles. EVs influence the behaviour of target cells in multiple ways, including the transfer of bioactive cargoes and the activation of signalling events at the cell surface (Holm et al., 2018). In addition, EVs possess enzymatic activity and are capable of modifying

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Section: Resultsmentioning

confidence: 97%

Astrocytes‐derived extracellular vesicles in motion at the neuron surface: Involvement of the prion protein

D’Arrigo

Gabrielli

Scaroni

et al. 2021

J of Extracellular Vesicle

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“…Cell membrane fluctuation analyses showed high CMF similarities between nRBC, nRET and cRBC lipids , while CMF is different for cRBC w/o lipids . However, this value is affected by the spherical shape of the cell and its height, as discussed in our recent publication (Bernecker et al, 2021).…”

Section: Discussionmentioning

confidence: 94%

“…Cell morphology and cell membrane fluctuations (CMF) were measured for RBC in physiological solution, with the cells placed on a coverslip, using a custom DHM system based on a Mach-Zehnder interferometer ( Bernecker et al, 2021 ). The morphological parameters: area, volume and sphericity were calculated from the cell height map as described ( Bernecker et al, 2021 ).…”

Section: Methodsmentioning

confidence: 99%

“…New biomechanical studies mainly focus on mature nRBC, while comprehensive investigations on native reticulocytes (nRET) and cRBC are scarce. Furthermore, recent data showed that one technique is not able to cover the variety of cells in shape, functionality and structure ( Bernecker et al, 2021 ). Each method has its limitations, and only a combination would allow the goal of an all-encompassing characterization to be achieved.…”

Section: Introductionmentioning

confidence: 99%

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Biomechanical properties of native and cultured red blood cells–Interplay of shape, structure and biomechanics

et al. 2022

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Modern medicine increases the demand for safe blood products. Ex vivo cultured red blood cells (cRBC) are eagerly awaited as a standardized, safe source of RBC. Established culture models still lack the terminal cytoskeletal remodeling from reticulocyte to erythrocyte with changes in the biomechanical properties and interacts with membrane stiffness, viscosity of the cytoplasm and the cytoskeletal network. Comprehensive data on the biomechanical properties of cRBC are needed to take the last step towards translation into clinical use in transfusion medicine. Aim of the study was the comparative analysis of topographical and biomechanical properties of cRBC, generated from human CD34+ adult hematopoietic stem/progenitor cells, with native reticulocytes (nRET) and erythrocytes (nRBC) using cell biological and biomechanical technologies. To gain the desired all-encompassing information, a single method was unsatisfactory and only the combination of different methods could lead to the goal. Topographical information was matched with biomechanical data from optical tweezers (OT), atomic force microscopy (AFM) and digital holographic microscopy (DHM). Underlying structures were investigated in detail. Imaging, deformability and recovery time showed a high similarity between cRBC and nRBC. Young’s modulus and plasticity index also confirmed this similarity. No significant differences in membrane and cytoskeletal proteins were found, while lipid deficiency resulted in spherical, vesiculated cells with impaired biomechanical functionality. The combination of techniques has proven successful and experiments underscore a close relationship between lipid content, shape and biomechanical functionality of RBC.

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“…Such a combination will allow investigating the morphological changes occurring to bio-samples in their natural environment, obtaining sample features otherwise inaccessible using optical tweezers, like sample volume, surface area, shape, and thickness fluctuations. Two-beam interferometer geometry has been reported for combining optical tweezers and digital holographic microscopy 25,[43][44][45] .…”

Section: Introductionmentioning

confidence: 99%

Integrated self-referencing single shot digital holographic microscope and optical tweezer

Utadiya¹,

Joshi²,

Patel³

et al. 2022

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Digital holographic microscopy is a single shot technique for quantitative phase imaging of objects, yielding thickness profiling of phase objects. It provides sample features based on their morphology, leading to their classification and identification. However, observing samples, especially cells, in fluids using holographic microscopes is difficult without immobilizing the object. Optical tweezers can be used for sample immobilization in fluids. The present manuscript provides an overview of our ongoing work on the development of a compact, low-cost microscopy system for digital holographic imaging of optically trapped samples. Integration of digital holographic microscopy system with tweezers is realized by using the optical pickup unit extracted from DVD burners to trap microsamples, which are then holographically imaged using a highly compact self-referencing interferometer along with a low-cost, in-house developed quadrant photodiode, providing morphological and spectral information of trapped particles. The developed integrated module was tested using polystyrene microspheres as well as human erythrocytes. The investigated system offers a multitude of sample features, including physical and mechanical parameters and corner frequency information of the sample. These features were used for sample classification. The proposed technique has vast potential in opening up new avenues for low-cost, digital holographic imaging and analysis of immobilized samples in fluids and their classification.

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Biomechanics of Ex Vivo-Generated Red Blood Cells Investigated by Optical Tweezers and Digital Holographic Microscopy

Cited by 15 publications

References 52 publications

Astrocytes‐derived extracellular vesicles in motion at the neuron surface: Involvement of the prion protein

Astrocytes‐derived extracellular vesicles in motion at the neuron surface: Involvement of the prion protein

Biomechanical properties of native and cultured red blood cells–Interplay of shape, structure and biomechanics

Integrated self-referencing single shot digital holographic microscope and optical tweezer

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