Compact microscope-based optical tweezers system for molecular manipulation

Abstract

A compact single beam optical tweezers system for force measurements and manipulation of individual double-stranded deoxyribonucleic acid ͑DNA͒ molecules was integrated into a commercial inverted optical microscope. A maximal force of 150 pN combined with a force sensitivity of less than 0.5 pN allows measurements of elastic properties of single molecules which complements and overlaps the force regime accessible with atomic force microscopy ͑AFM͒. The manipulation and measurement performance of this system was tested with individual -DNA molecules and renders new aspects of dynamic forces phenomena with higher precision in contrast to AFM studies. An integrated liquid handling system with a fluid cell allows investigation of the force response of individual DNA molecules in the presence of DNA binding agents. Comparison of YOYO-1-, ethidium bromide intercalated DNA, and distamycin-A complexed DNA revealed accurate and reproducible differences in the force response to an external load. This opens the possibility to use it as a single molecule biosensor to investigate DNA binding agents and even to identify molecular binding mechanisms.

Bioanalysis in structured microfluidic systems

“…Individual Sf9 cells were trapped, injected and steered in the microfluidic channel by an in-house-built single-beam optical tweezers (OT) system which was incorporated into an inverted optical microscope [28] with additional standard fluorescence microscopy capabilities in the visible range. A selfconstructed x/y-stage allowed longrange positioning of cells with the OT within the microchannel for maximal 25 mm with a precision of 1-2 mm.…”

Single cell manipulation, analytics, and label-free protein detection in microfluidic devices for systems nanobiology

“…The OT was incorporated into an inverted optical microscope [29] with additional standard fluorescence microscopy capabilities in the VIS range. A selfconstructed x/y-stage allowed long range positioning of cells with the OTs within the microchannel for maximal 25 mm with a precision of 1-2 mm.…”

Single cell analytics for nanobiology

“…Manipulation and steering of micron-sized objects like beads, colloids and cells is possible with a spatial resolution of 1 nm, a force sensitivity of 0.1 pN and a maximum force of up to 300 pN. Instrumental details have been published recently [7]. Living chicken DT40 B-cells were cultured in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, 1% heat-inacitivated chicken serum, 10 units/ml penicillin, 10 µg/ml streptomycin and 2 mM L-glutamine at 37°C and 5% CO 2 .…”

“…The detection and analysis of single biomolecules, of smallest analyte quantities and the hunt for low abundant proteins at the single cell level, however, require new sensitive and efficient techniques. In systems nanobiology [2], microfabrication and nanotechnology offer novel tools to detect, image, measure, analyze, steer, and manipulate individual molecules and cells by AFM [3][4][5][6] and OT [7] and will allow more detailed insights into the physical mechanism of specific interaction, binding kinetics, and the interplay of genomic information and functional peculiarity at the single molecule or single cellular level. Micro total analysis systems (µTAS) or lab-on-a-chip systems [8,9] offer the possibility to handle minute volumes down to the pL and even fL range.…”