High-Resolution Optical Tweezers Combined With Single-Molecule Confocal Microscopy.

We describe the design, construction, and application of an instrument combining dual-trap, high-resolution optical tweezers and a confocal microscope. This hybrid instrument allows nanomechanical manipulation and measurement simultaneously with single-molecule fluorescence detection. We present the general design principles that overcome the challenges of maximizing optical trap resolution while maintaining single-molecule fluorescence sensitivity, and provide details on the construction and alignment of the instrument. This powerful new tool is just beginning to be applied to biological problems. We present step-by-step instructions on an application of this technique that highlights the instrument's capabilities, detecting conformational dynamics in a nucleic acid-processing enzyme.

Ultrasensitive magnetic biosensor for homogeneous immunoassay.

A technique is described for specific, sensitive, quantitative, and rapid detection of biological targets by using superparamagnetic nanoparticles and a "microscope" based on a high-transition temperature dc superconducting quantum interference device (SQUID). In this technique, a mylar film to which the targets have been bound is placed on the microscope. The film, at room temperature and atmospheric pressure, is typically 40 micrometer from the SQUID, which is at 77 K in a vacuum. A suspension of magnetic nanoparticles carrying antibodies directed against the target is added to the mixture in the well, and 1-s pulses of magnetic field are applied parallel to the SQUID. In the presence of this aligning field the nanoparticles develop a net magnetization, which relaxes when the field is turned off. Unbound nanoparticles relax rapidly by Brownian rotation and contribute no measurable signal. Nanoparticles that are bound to the target on the film are immobilized and undergo Néel relaxation, producing a slowly decaying magnetic flux, which is detected by the SQUID. The ability to distinguish between bound and unbound labels allows one to run homogeneous assays, which do not require separation and removal of unbound magnetic particles. The technique has been demonstrated with a model system of liposomes carrying the FLAG epitope. The SQUID microscope requires no more than (5 +/- 2) x 10(4) magnetic nanoparticles to register a reproducible signal.

A new study of bacterial motion: superconducting quantum interference device microscopy of magnetotactic bacteria.

The recently developed "microscope" based on a high-Tc dc SQUID (superconducting quantum interference device) is used to detect the magnetic fields produced by the motion of magnetotactic bacteria, which have permanent dipole moments. The bacteria, in growth medium at room temperature, can be brought to within 15 micron of a SQUID at liquid nitrogen temperature. Measurements are performed on both motile and nonmotile bacteria. In the nonmotile case, we obtain the power spectrum of the magnetic field noise produced by the rotational Brownian motion of the ensemble of bacteria. Furthermore, we measure the time-dependent field produced by the ensemble in response to an applied uniform magnetic field. In the motile case, we obtain the magnetic field power spectra produced by the swimming bacteria. Combined, these measurements determine the average rotational drag coefficient, magnetic moment, and the frequency and amplitude of the vibrational and rotational modes of the bacteria in a unified set of measurements. In addition, the microscope can easily resolve the motion of a single bacterium. This technique can be extended to any cell to which a magnetic tag can be attached.