Force measurements made with a translating holographic optical trap (HOT) of a viscous and a viscoelastic medium are
investigated. In purely viscous media, Stokes drag cannot be measured with a translating HOT with established methods.
In the viscoelastic system of the pericellular coat, the standard force curves generated by a fixed optical trap coupled with
a moving stage can reliably be reproduced by translating HOT experiments. The viscoelastic cell coat provides an
example where slow relaxation dynamics makes force measurements relatively insensitive to differences between
measurements. These preliminary studies suggest that when the relaxation time scale of a system is much slower than
the time scale of the HOT updates, translating HOTs can be reliably used to make force measurements on a viscoelastic, non-equilibrium system.
The actin cortex is an adaptive chemo-mechanical polymer network located beneath the cell membrane. A thin, quasi two-dimensional (2D) network, the actin cortex plays a leading role in controlling cellular viscoelasticity, shape, and motility. Regulated by internal and external stimuli, the actin cortex varies its properties with controlled polymerization and depolymerization of actin. For constructing and probing biomimetic actin networks we combined three different techniques to achieve complete spatial, visual and chemical control of the microenvironment: 1) dynamic holographic optical tweezers (HOTs) which produce and independently steer one to hundreds of optical traps, 2) fluorescence microscopy for imaging of actin and 3) a specially-designed microfluidic system, which precisely controls the chemical environment. Using this system, we take two approaches to construct biomimetic 2D actin networks. First HOTs micropattern surfaces with microspheres onto which actin can the be grown. Secondly, HOTs in combination with a multi channel microfluidic system are used to coat optically-trapped microsphere arrays with actin.
Holographic optical tweezers (HOTs) techniques are further developed to study hyaluronan-mediated adhesion of chondrocyte cells. We present a calibration scheme and address fundamental issues concerning the use of HOTs for quantitative force measurements. Influence of SLM pixelation on trap stiffness is observed and can be utilized to design calibrated HOTs more effectively. It is also shown that the HOTs trapping stiffness can vary significantly over short distances. Then we use HOTs cell adhesion assays investigate the viscoelastic and adhesive nature of chondrocytes' pericellular matrix (PCM) at two different time steps (30 minute and 24 hour incubation periods). Surprisingly, no physical influence of the large, presumably gel-like PCM is observed. However, a difference is discerned in the adhesiveness of the two sets of cells. The early-stage cells have reversible adhesion with negatively-charged and fibronectin-coated microspheres even after they are held at the cell surface for 10 seconds. In contrast, late stage cells stick irreversibly to all types of beads: positive, negative, fibronectin and hyaluronan-coated. Additionally, only the late stage cells produce membrane tethers. These observations suggest that the late-stage chondrocytes have less surface-associated hyaluronan and have interesting implications for the role of hyaluronan in the early stages of cell adhesion.
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