An integrated laser trap/flow control video microscope for the study of single biomolecules.
We have developed an integrated laser trap/flow control video microscope for mechanical manipulation of single biopolymers. The instrument is automated to maximize experimental throughput. A single-beam optical trap capable of trapping micron-scale polystyrene beads in the middle of a 200-microm-deep microchamber is used, making it possible to insert a micropipette inside this chamber to hold a second bead by suction. Together, these beads function as easily exchangeable surfaces between which macromolecules of interest can be attached. A computer-controlled flow system is used to exchange the liquid in the chamber and to establish a flow rate with high precision. The flow and the optical trap can be used to exert forces on the beads, the displacements of which can be measured either by video microscopy or by laser deflection. To test the performance of this instrument, individual biotinylated DNA molecules were assembled between two streptavidin beads, and the DNA elasticity was characterized using both laser trap and flow forces. DNA extension under varying forces was measured by video microscopy. The combination of the flow system and video microscopy is a versatile design that is particularly useful for the study of systems susceptible to laser-induced damage. This capability was demonstrated by following the translocation of transcribing RNA polymerase up to 650 s.
Wuite
G J
GJ
Department of Physics, University of California, Berkeley, California 94720 USA.
Davenport
R J
RJ
Rappaport
A
A
Bustamante
C
C
D002138:Calibration; D004247:DNA / Q000737:chemistry* / Q000648:ultrastructure; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000648:ultrastructure; D004548:Elasticity; D004867:Equipment Design; D007834:Lasers; D018715:Microscopy, Video / Q000295:instrumentation* / Q000379:methods*; D009690:Nucleic Acid Conformation; D055095:Optics and Photonics
D004247:DNA; D012321:DNA-Directed RNA Polymerases