Jason Fisher

Thin-foil magnetic force system for high-numerical-aperture microscopy

Forces play a key role in a wide range of biological phenomena from single-protein conformational dynamics to transcription and cell division, to name a few. The majority of existing microbiological force application methods can be divided into two categories: those that can apply relatively high forces through the use of a physical connection to a probe and those that apply smaller forces with a detached probe. Existing magnetic manipulators utilizing high fields and high field gradients have been able to reduce this gap in maximum applicable force, but the size of such devices has limited their use in applications where high force and high-numerical-aperture (NA) microscopy must be combined. We have developed a magnetic manipulation system that is capable of applying forces in excess of 700 pN on a 1 mum paramagnetic particle and 13 nN on a 4.5 mum paramagnetic particle, forces over the full 4pi sr, and a bandwidth in excess of 3 kHz while remaining compatible with a commercially available high-NA microscope objective. Our system design separates the pole tips from the flux coils so that the magnetic-field geometry at the sample is determined by removable thin-foil pole plates, allowing easy change from experiment to experiment. In addition, we have combined the magnetic manipulator with a feedback-enhanced, high-resolution (2.4 nm), high-bandwidth (10 kHz), long-range (100 mum xyz range) laser tracking system. We demonstrate the usefulness of this system in a study of the role of forces in higher-order chromosome structure and function.

Three-dimensional force microscope: A nanometric optical tracking and magnetic manipulation system for the biomedical sciences

We report here the development of a three-dimensional (3D) magnetic force microscope for applying forces to and measuring responses of biological systems and materials. This instrument combines a conventional optical microscope with a free-floating or specifically bound magnetic bead used as a mechanical probe. Forces can be applied by the bead to microscopic structures of interest (specimens), while the reaction displacement of the bead is measured. This enables 3D mechanical manipulations and measurements to be performed on specimens in fluids. Force is generated by the magnetically permeable bead in reaction to fields produced by external electromagnets. The displacement is measured by interferometry using forward light scattered by the bead from a focused laser beam. The far-field interference pattern is imaged on a quadrant photodetector from which the 3D displacement can be computed over a limited range about the focal point. The bead and specimen are mounted on a 3D translation stage and feedback t...

DNA relaxation dynamics as a probe for the intracellular environment

Investigations into the biophysical properties of single molecules traditionally involve well defined in vitro systems where parameters such as solvent viscosity and applied forces are known a priori. These systems provide means to develop models describing the polymers response to a variety of conditions, including the entropically driven relaxation of a stretched biopolymer upon release of the tension inducing force. While these techniques have proven instrumental for recent advancements in the fields of polymer physics and biophysics, how applicable they are to life inside the cell remains poorly understood. Here we report an investigation of in vivo stretched polymer relaxation dynamics using chromatin relaxation following the breakage of a dicentric chromosome subjected to microtubule-based spindle forces. Additionally, we have developed an in vitro system used to verify the conformations observed during the in vivo relaxation, including the predicted but previously unidentified taut conformation. These observations motivate our use of existing polymer models to determine both the in vivo viscosity as seen by the relaxing chromatin and the tension force applied by the microtubule-based spindle in vivo. As a result, the technique described herein may be used as a biophysical strategy to probe the intranuclear environment.

MAGNETIC FORCE MICROMANIPULATION SYSTEMS FOR THE BIOLOGICAL SCIENCES

Manipulation systems using magnetic field gradients have the ability to apply a large range of forces noninvasively to a specific target. Depending on the requirements of a given experiment, the systems may be as simple as a single electromagnet for unidirectional manipulation or as complex as a high-frequency three-dimensional manipulator with force feedback. Here, we discuss the motivation for developing such systems, theory and design considerations, and give examples of the broad range of manipulators that has been put to use. In addition, we discuss a variety of applications demonstrating the range of experiments for which such a system is applicable.

The software interface to the 3D-force microscope

We have developed a real-time experiment-control and data-display system for a novel microscope, the 3D-force microscope (3DFM), which is designed for nanometer-scale and nanoNewton-force biophysical experiments. The 3DFM software suite synthesizes the several data sources from the 3DFM into a coherent view and provides control over data collection and specimen manipulation. Herein, we describe the system architecture designed to handle the several feedback loops and data flows present in the microscope and its control system. We describe the visualization techniques used in the 3DFM software suite, where used, and on which types of data. We present feedback from our scientist-users regarding the usefulness of these techniques, and we also present lessons learned from our successive implementations.