Feasibility Studies for Optical Trap Detection of Single Molecule Adhesion and Force Generation

Open Access
Author:
Bernick, David J
Area of Honors:
Bioengineering
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Peter J Butler, Thesis Supervisor
  • Peter J Butler, Honors Advisor
  • Keefe B Manning, Faculty Reader
  • William O Hancock, Faculty Reader
Keywords:
  • optical trap
  • laser tweezers
  • integrin ligand binding
  • bead
  • avidin
  • biotin
  • microscopy
Abstract:
The optical trap (laser tweezers) is an instrument which uses laser radiation and the focusing power of a microscope to trap microscopic particles such as beads, bacteria, and organelles. The particles are pulled into the focal point of the trap and held in three dimensions by the transfer of the photons’ momentum. The resultant force (F) can be modeled as a Hookean spring, F = kspring x with the forces used to hold the particle dependent on the particle’s displacement (x) from the trap center. Optical traps and can measure forces at the piconewton level. It has been reported that during binding reactions between receptors and ligands, piconewton forces and nanometer displacements are produced on time scales that are on the order of micro- to milliseconds. Thus, an optical trap is an ideal instrument to detect these events as they happen, provided the position detection can be accomplished at these very short time scales. Previously, our optical trap measured displacements using a CCD camera followed by displacement assessment using image correlation-based tracking. This method had a disadvantage in that it could only measure positions up to about 50 Hz and required simultaneous differential interference contrast imaging of the bead. The requirement for imaging can be a disadvantage when it is desired to use the microscope to detect other events based on fluorescence. The focus of this study was to integrate a Quadrant Photodiode (QPD) into a conjugate focal plane behind the condenser. A QPD, when properly aligned and calibrated, can detect the changes in laser beam position caused by small movement of the bead in the trap. The update rate of the QPD is about 30 kHz allowing three dimensional tracking of bead position of greater than 10 kHz using an analog to digital converter. Thus the main aims of this research were to (i) determine the range, measured from the coverslip surface, within which the kspring is constant, (ii) determine the relationship between QPD voltage and bead displacement from the trap center, (iii) assess the feasibility of measuring rapid binding events, and (iv) conduct preliminary studies on the detection of binding events between a fibronectin functionalized bead and integrin receptors on the endothelial cell surface.