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

Ex vivo-generated red blood cells are a promising resource for future safe blood products, manufactured independently of voluntary blood donations. The physiological process of terminal maturation from spheroid reticulocytes to biconcave erythrocytes has not been accomplished yet. A better biomechanical characterization of cultured red blood cells (cRBCs) will be of utmost interest for manufacturer approval and therapeutic application. Here, we introduce a novel optical tweezer (OT) approach to measure the deformation and elasticity of single cells trapped away from the coverslip. To investigate membrane properties dependent on membrane lipid content, two culture conditions of cRBCs were investigated, cRBCPlasma with plasma and cRBCHPL supplemented with human platelet lysate. Biomechanical characterization of cells under optical forces proves the similar features of native RBCs and cRBCHPL, and different characteristics for cRBCPlasma. To confirm these results, we also applied a second technique, digital holographic microscopy (DHM), for cells laid on the surface. OT and DHM provided related results in terms of cell deformation and membrane fluctuations, allowing a reliable discrimination between cultured and native red blood cells. The two techniques are compared and discussed in terms of application and complementarity.

Probing Cell Mechanics with Bead-Free Optical Tweezers in the Drosophila Embryo.

Morphogenesis requires coordination between genetic patterning and mechanical forces to robustly shape the cells and tissues. Hence, a challenge to understand morphogenetic processes is to directly measure cellular forces and mechanical properties in vivo during embryogenesis. Here, we present a setup of optical tweezers coupled to a light sheet microscope, which allows to directly apply forces on cell-cell contacts of the early Drosophila embryo, while imaging at a speed of several frames per second. This technique has the advantage that it does not require the injection of beads into the embryo, usually used as intermediate probes on which optical forces are exerted. We detail step by step the implementation of the setup, and propose tools to extract mechanical information from the experiments. By monitoring the displacements of cell-cell contacts in real time, one can perform tension measurements and investigate cell contacts' rheology.

Biolens behavior of RBCs under optically-induced mechanical stress.

In this work, the optical behavior of Red Blood Cells (RBCs) under an optically-induced mechanical stress was studied. Exploiting the new findings concerning the optical lens-like behavior of RBCs, the variations of the wavefront refracted by optically-deformed RBCs were further investigated. Experimental analysis have been performed through the combination of digital holography and numerical analysis based on Zernike polynomials, while the biological lens is deformed under the action of multiple dynamic optical tweezers. Detailed wavefront analysis provides comprehensive information about the aberrations induced by the applied mechanical stress. By this approach it was shown that the optical properties of RBCs in their discocyte form can be affected in a different way depending on the geometry of the deformation. In analogy to classical optical testing procedures, optical parameters can be correlated to a particular mechanical deformation. This could open new routes for analyzing cell elasticity by examining optical parameters instead of direct but with low resolution strain analysis, thanks to the high sensitivity of the interferometric tool. Future application of this approach could lead to early detection and diagnosis of blood diseases through a single-step wavefront analysis for evaluating different cells elasticity. © 2017 International Society for Advancement of Cytometry.

Potentialities of laser trapping and manipulation of blood cells in hemorheologic research.

Laser trapping and manipulation of blood cells without mechanical contact have become feasible with implication of laser tweezers. They open up new horizons for the hemorheologic researches, offer new possibilities for studying live cells interactions on individual cell level under the influence of different endogenous and exogenous factors. The operation principle of laser tweezers is based on the property of strongly focused laser beam to act on a dielectric microparticle located in the vicinity of the beam waist with a force that drives the particle to the equilibrium location and holds it there. If the beam waist position is manipulated, so is the position of the particle. The displacement of the particle from the equilibrium position by external forces can be calibrated so that these forces can be precisely measured in the range ca. 0.1-100 pN. This is the range of forces of elastic deformation of blood cells and of their interaction with each other and with vessel walls. Being able to measure these forces without mechanical contact allows for studying on single cell level the mechanisms of interactions that was impossible earlier. Here we discuss the basic features of these techniques and give some examples of challenging hemorheologic studies.

Yoctoliter thermometry for single-molecule investigations: a generic bead-on-a-tip temperature-control module.

A new temperature-jump (T-jump) strategy avoids photo-damage of individual molecules by focusing a low-intensity laser on a black microparticle at the tip of a capillary. The black particle produces an efficient photothermal effect that enables a wide selection of lasers with powers in the milliwatt range to achieve a T-jump of 65 °C within milliseconds. To measure the temperature in situ in single-molecule experiments, the temperature-dependent mechanical unfolding of a single DNA hairpin molecule was monitored by optical tweezers within a yoctoliter volume. Using this bead-on-a-tip module and the robust single-molecule thermometer, full thermodynamic landscapes for the unfolding of this DNA hairpin were retrieved. These approaches are likely to provide powerful tools for the microanalytical investigation of dynamic processes with a combination of T-jump and single-molecule techniques.

Optical-tweezer-induced microbubbles as scavengers of carbon nanotubes.

A modified optical tweezers set-up has been used to generate microbubbles in flowing, biologically relevant fluids and human whole blood that contains carbon nanotubes (CNTs) using low power (< or =5 mW), infrared (1064 nm wavelength), continuous wave laser light. Temperature driven effects at the tweezers' focal point help to optically trap these microbubbles. It is observed that proximate CNTs are driven towards the focal spot where, on encountering the microbubble, they adhere to it. Such CNT-loaded microbubbles can be transported both along and against the flow of surrounding fluid, and can also be exploded to cause fragmentation of the bundles. Thus, microbubbles may be used for scavenging, transporting and dispersal of potentially toxic CNTs in biologically relevant environments.