DEVELOPMENT OF A LASER TRAP BASED MICRO-MECHANICAL FORCE MANIPULATION AND MEASUREMENT WORKSTATION WITH COMBINED LOW LIGHT LEVEL FLUORESCENCE AND TRANSMITTED LIGHT IMAGING

RW Stroetz, BJ Walters with the guidance of William H Guilford (UVA)
Optical traps (tweezers) are based on the theory of radiation pressure. By using the various effects of light, dielectric particles ranging in size from sub micron to hundreds of microns can be successfully held by a beam (or beams) of light. Initially developed at Bell Labs 1986 (Ashkin), optical tweezers are primarily used for manipulating components of biological cells. A variant of the system has also been used to slow and trap individual atoms.

Motivation
The development of optical tweezers by this lab as a tool to manipulate microscopic particles began with the desire to manipulate single cells as well as sub cellular structures. Because optical traps behave as linear springs, optical tweezers can serve as an ideal force transducer when coupled with a sensitive position detection system. A trap exerts a linear restoring force proportional to trap stiffness. Innovations in tweezer design now give optical tweezers the advantages of computer control and feedback between detection and positioning. Using optical tweezers and related optical technology, one can apply pico-newton sized loads and measure nanometer level displacements.

Light photons have mass & inertia. Trapping forces originate from radiation pressure. Cartoon shows a high refractive index bead placed off axis and above focal point of a focused Gaussian laser beam. Considering two typical ray paths “A” and “B” striking the bead on opposing surfaces, refraction through bead (as A & B change path) gives rise to forces FA & FB. Since light intensity is greater at center, FB > FA and bead is first pushed to center of beam in X and Y and then to focal point in Z axis as forces equilibrate about center of bead (conservation of momentum)

Cell membranes may be grasped and deformed. Series at left shows lipid tethor formation of cell membrane as ligand coated bead is first attached to cell membrane, then trapped by laser. Tethor is formed when bead is pulled downward in direction of red arrows.

In short, mechanical forces have long been shown to have powerful influences on the behavior of cells in health and disease. The laser tweezers system described here will be used impart mechanical perturbations on a nanometer scale, measure mechanical forces on a pico-Newton scale, and study the resulting biology in a variety of pulmonary cells in culture, specifically to probe the physics of alveolar epithelial membrane. The tweezers will be capable of not only measuring membrane properties such as stiffness and yield strength, but also be capable of manipulating sub cellular structures such as focal adhesion complexes through the creation of multiple traps. The laser tweezer unit is part of an inverted microscope system which retains transmitted and multi channel fluorescence capabilities and as such allows observation of biological events related to the physical manipulations imparted by the laser.

COMPUTER CONTROLLED CELL STRAIN UNIT

RW Stroetz, BJ Walters
Motivated by our interest in lung deformation injury, we have developed a new live cell strain system, shown below. Cells are grown on flexible membranes to which they attach and are shown in the figure below, upper left. The system allows microscopic imaging while the cell culture environment is maintained equivalent to that provided by conventional incubators. The straining action occurs in direct proportion to the vertical displacement of diaphragm and piston, shown in the cartoon lower left. This mechanical action is based on two computer controlled linear actuators which operate synchronously in closed loop mode. The cycling amplitude, cycling rate and straining profile are programmable in similar fashion to a mechanical ventilator. The strain ouput is uniform and reproducible. Using this system we defined cell deformation dose (i.e. membrane strain amplitude)-cell injury response relationships in alveolar epithelial cultures and studied the effects of temperature on these. Deformation injury occurred in the form of reversible, non-lethal plasma membrane stress failure events and was quantified using a variety of fluoescent imaging techniques.