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CISMM is measuring the forces exerted by cilia in human lung cell cultures to understand mucus clearance. A magnetic bead attached to a cilia can permit 3D laser tracking of cilia trajectory and apply forces to measure mechanoresponse of epithelial layer. (Collaborator: R. Boucher - UNC Cystic Fibrosis Center).

Computer Integrated Systems for Microscopy and Manipulation

Contents

• Contact Information
• Grant Number
• Research Emphasis
• Current Research
• Resource Capabilities
• References

Contact Information

Department of Physics and Astronomy
Phillips Hall CB3255
University of North Carolina
Chapel Hill, NC 27599-3255
http://www.cs.unc.edu/Research/nano/cismm/index.html

Principal Investigator/Contact
Richard Superfine, Ph.D.
Phone: 919-962-1185
rsuper@physics.unc.edu

Contact
Maggie Hudson
Phone: 919-962-4057
Fax: 919-962-0480
maggie@physics.unc.edu

Grant Number

Grant No. EB002025

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Research Emphasis

Our Force Microscope Technologies Core designs instruments in an area of science where there are unusual opportunities: the measurement of forces and the integration with optical microscopy. Force technologies play the obvious role of both measuring events in the sample and modifying the sample during the experiment. It is through the microscope that the force data is correlated with simultaneous 3D optical images. The force technology development includes the magnetic bead technology in our 3D Force Microscope project, Atomic Force Microscopy in our nanoManipulator project, and Control Software to drive the instrumentation. This core is focused on providing the physical capability to perform the experiments and probe structure/property correlations.

The Ideal User Interfaces core makes the connection between the user and the instrument, the model building, and the data. This includes control systems that allow the user to move the bead inside the cell culture with a handheld pen and the visualization techniques to view the optical microscope data as a rendered 3D image collocated with the force data.

Using data to create, change, and understand a model is the focus of our Advanced Model Fitting and Analysis core. The quantitative reduction of images to structural, shape, and velocity parameters is the goal of Image Analysis. The immediate understanding of correlations across image fields and between data sets in the challenge of Visualization. The power of combining the strength of a computer science graphics group with a microscopy technology group is most evident in the Graphics Hardware Acceleration project, which seeks to harness the speed of graphics processors for microscope data analysis and simulation.

Our Advanced Technology core pushes the boundaries of the Human Computer Interface through the investigation of improved techniques for the interaction of users with virtual environments, the real time lighting of virtual settings, and the enabling of multi-person collaboration. These techniques are validated and evaluated through physiological measures in virtual environments effectiveness evaluation studies.

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Current Research

Our collaborations span the range from single molecule studies to the physiological implications of cell culture studies for healthy pulmonary function. At the smallest scale our collaborators are studying the force response of chromatin and the role this response may play in gene expression and cell cycle regulation. This is accomplished by pulling on beads attached to single strands of chromatin and observing 50 nm steps as nucleosomes release under load. Forces inside the dividing mitotic spindle are studied in experiments using magnetic beads as model kinetochores. The response of the spindle to force is recorded in 3d in our combined magnetic force/confocal microscope system.

We are measuring the mechanical properties of fibrin fibers to understand the implications of individual protein assemblies on clotting and bleeding disorders. Individual quantum dot labeled fibers are manipulated using an Atomic Force Microscope while observing strain in real time in the optical microscope.

At the cell culture level, we are measuring the forces exerted by cilia in human lung cell cultures, and measuring the response of the mucus overlayer. This will allow the UNC Cystic Fibrosis Center to understand the genetic origins of the failure of mucus clearance, and design effective therapies to restore healthy function.

Our visualization and analysis tools are helping to guide surgery through effective segmentation of MRI for tumor surgery. The stabilization of images to understand time series in 3D structural images from confocal microscopy is being applied to microtubule fluxing during cell division. Understanding multiple parameter 3D data sets remains a difficult challenge. Our 3D visualization and analytical software is being used to understand the role of dendritic spines as potential signaling sites on the neuron. Confocal data sets imaging multiple messengers are analyzed quantitatively to understand correlated signaling candidates on the spine.

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Resource Capabilities

Instruments

• Veeco Explorer Atomic Force Microscope
• Veeco Explorer Atomic Force Microscope on a Nikon Diaphot200 fluorescence microscope
• Thermomicroscopes Discoverer Atomic Force microscope
• Hitachi S4700 Scanning Electron Microscope
• CISMM 3D Force Microscope Magnetics system on Nikon TE2000 Fluorescence Microscope
• CISMM 3DFM on Nikon TE2000 Fluorescence Microscope equipped with McBain Instruments Spinning Disk Confocal System.
• Cell Culture Facility with laminar flow hood, -80 freezer and incubator.
• Sensable Technologies Phantom force-feeedback devices for microscope control (4)

Software

• ImageSurfer is a software application used to view stacks of grayscale images, taken from a confocal microscope, as volumes.
• The Video Spot Tracker program is used to track the motion of one or more spots in a Microsoft DirectShow-compatible video file, from a Microsoft DirectShow-compatible camera, from a Roper Scientific camera, or from a DiagInc SPOT camera.
• The AFM Simulator is an application written using Java and the open source Visualization Toolkit. The application enables a scientist to construct a 3D scene from primitives or imported files such Wavefront .OBJ or .PDB files from the Protein Databank. Once a 3D model has been created, the user can specify the parameters of a simple AFM tip model and quickly generate a simulated AFM surface scan.
• VRPN-CISMM developed a core library for interfacing with various virtual-reality devices (locally or across a network) that supports trackers from more than six different vendors and more than fourteen button/analog/joystick devices. The library, named the "Virtual Reality Peripheral Network," is written in C++ and has been ported to many variants of Linux and to Windows. There are Java bindings for the remote "user" objects (as opposed to server objects) in development.
• The Plot Extractor program is used to extract plot data from a TIFF image of a plot or series of plots. The data is saved in a format readable by Mathematica. The program currently runs only on Windows, but it uses the portable openGL library for rendering, the portable Tcl/Tk for user interface control, and the portable DevIL package for reading images.

Special Features

• Forces in Biology Short Course: Biomedical Engineering Society Annual Meeting 2004
• Force Measurement and Manipulation in Biological Microscopy, UNC-Chapel Hill, Summer 2005
• We support on-site visits of users from across the country, place hardware systems into collaborators laboratories, and distribute and maintain free software for the biomedical research community.

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References

1. Guthold M, Liu W, Stephens B, Lord ST, Hantgan RR, Erie DA, Taylor II RM, Superfine R. Visualization and mechanical manipulations of individual fibrin fibers. Biophys. J. 2004;87(6):4226-4236.
2. Negishi A, Chen J, McCarty D, Samulski RJ, Liu J, Superfine R. Analysis of the interaction of adeno-associated virus and heparan sulfate using atomic force microscopy. Glycobiology 2004;14(11):969-977.
3. Lok B, Naik S, Whitton MC, Brooks Jr. FP. Effects of handling real objects and self-avatar fidelity on cognitive task performance and sense of presence in virtual environments. Journal on Presence: Teleoperators and Virtual Environments 2004;12(6):615-628.
4. Taylor II RM, Borland D, Brooks Jr. FP, Falvo M, Guthold M, Hudson T, Jeffay K, Jones G, Marshburn D, Papadakis SJ, Qin L-C, Seeger A, Smith FD, Sonnenwald DH, Superfine R, Washburn S, Weigle C, Whitton MC, Williams P, Vicci L, Robinett W. Visualization and natural control systems for microscopy. In: Visualization Handbook. C. J. a. C. Hansen, Harcourt Academic Press, 2004. pp. 875-900.
5. Wang H, Yang Y, Schofield MJ, Du C, Fridman Y, Lee SD, Larson ED, Drummond DT, Alani E, Hsieh P, Erie D. DNA bending and unbending by MutS govern mismatch recognition and specificity. Proceedings of the National Academy of Sciences 2003;100:14822-14827.