An Integrated Laser Trap/Flow Control Video Microscope for the Study of Single Biomolecules

Department of Physics, University of California, Berkeley, California 94720 USA.
Biophysical Journal (Impact Factor: 3.97). 09/2000; 79(2):1155-67. DOI: 10.1016/S0006-3495(00)76369-7
Source: PubMed


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.

  • Source
    • "Ò (Wuite et al., 2000a "
    [Show abstract] [Hide abstract]
    ABSTRACT: The technically challenging field of single-molecule biophysics has established itself in the last decade by granting access to detailed information about the fate of individual biomolecules, unattainable in traditional biochemical assays. The appeal of single-molecule methods lies in the directness of the information obtained from individual biomolecules. Technological improvements in single-molecule methods have made it possible to combine optical tweezers, fluorescence microscopy, and microfluidic flow systems. Such a combination of techniques has opened new possibilities to study complex biochemical reactions on the single-molecule level. In this chapter, we provide general considerations for the development of a combined optical trapping, fluorescence microscopy, and microfluidics instrument, along with methods to solve technical issues that are critical for designing successful experiments. Finally, we present several experiments to illustrate the power of this combination of techniques.
    Full-text · Article · Jan 2010 · Methods in enzymology
  • Source
    • "Recently, several studies have focused on manipulation of DNA or physical properties of DNA by using different methods. Wuite et al. (2000) developed and integrated laser trap/flow control video microscope for mechanical manipulation of single biopolymers. For the purpose of understanding their traits, they studied the elasticity of DNA using the combined laminar/optical mode or the flow control system alone to exert the force. "
    [Show abstract] [Hide abstract]
    ABSTRACT: This research develops a bio-nanotechnology for mechanically handling individual DNA fibers. The current study particularly focuses on surgery of a chromosome, which is a long strand of DNA tightly wound on proteins. Since DNA wound on a chromosome is several centimeter long, it is too long to observe under a microscope. Therefore, it is necessary to develop a method for partial unwinding. This work uses laser-induced local heating. Focusing a laser beam onto an aimed location on a chromosome immersed in protease solution, induces temperature to rise and locally activates the enzymatic reaction. This work demonstrates stretching DNA fibers out of a chromosome using electro-osmotic flow. Results show a chromosome immobilized onto a glass surface with released DNA fibers as long as 150μm.
    Full-text · Article · Aug 2009 · Journal of the Chinese Society of Mechanical Engineers, Transactions of the Chinese Institute of Engineers - Series C
  • Source
    • "Recently, our group has demonstrated the feasibility of simultaneously performing controlled force-extension measurements and sensitive fluorescence detection in wide-field epi-fluorescence mode. The use of a water-immersion objective allowed for stable trapping even at a significant distance of the captured DNA from the glass surfaces of the sample chamber (41,49), which reduced background fluorescence without the need for any surface blocking. We applied this to the study of RAD51 filament mechanics on double-stranded DNA (50) (Figure 4B). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Direct visualization of DNA and proteins allows researchers to investigate DNA–protein interactions with great detail. Much progress has been made in this area as a result of increasingly sensitive single-molecule fluorescence techniques. At the same time, methods that control the conformation of DNA molecules have been improving constantly. The combination of both techniques has appealed to researchers ever since single-molecule measurements have become possible and indeed first implementations of such combined approaches have proven useful in the study of several DNA-binding proteins in real time. Here, we describe the technical state-of-the-art of various integrated manipulation-and-visualization methods. We first discuss methods that allow only little control over the DNA conformation, such as DNA combing. We then describe DNA flow-stretching approaches that allow more control, and end with the full control on position and extension obtained by manipulating DNA with optical tweezers. The advantages and limitations of the various techniques are discussed, as well as several examples of applications to biophysical or biochemical questions. We conclude with an outlook describing potential future technical developments in combining fluorescence microscopy with DNA micromanipulation technology.
    Full-text · Article · Jul 2008 · Nucleic Acids Research
Show more