How do you examine an unfamiliar object? You look at it. When possible, you pick it up, hold it at arm’s length, and turn it around. You may squeeze or prod to determine its stiffness; a fingernail feels for grooves or surface texture. If the object is on a surface, you may use a fingertip or pen to roll it around.How do you examine an unfamiliar object? You look at it. When possible, you pick it up, hold it at arm’s length, and turn it around. You may squeeze or prod to determine its stiffness; a fingernail feels for grooves or surface texture. If the object is on a surface, you may use a fingertip or pen to roll it around.
The nanoManipulator (nM) system provides a scientist with the ability to perform these actions on objects as small as single molecules while quantitatively measuring both the surface shape and forces applied. The nM uses the ultra-sharp tip of an atomic-force microscope (AFM) as tool both to scan and to modify samples. It uses advanced computer graphics to display the scanned surface to the user. A robot arm enables the user to feel and modify the surface (Taylor II 1993; Finch 1995; Taylor II 1997; Grant 1998; Taylor II 2003). — pic right of this paragraph!
The nM is the longest running of the current Resource projects, and is in the most advanced state of development. We continue to improve the system so that it remains useful in new experimental settings. The nM system currently has over 100 hierarchically grouped functions, each asked for by a user to address a particular challenge in an experiment. It has been ported from its initial configuration using a custom-built haptic display device, a custom-built SPM controller, and a custom-built graphics supercomputer to a configuration running entirely on commercially available equipment (PC-based graphics, the Phantom haptic display and Asylum SPMs).
New Direction: AFM/Fluorescence Correlation
Resource collaborator Ingrid Tessmer was studying DNA repair in Dorothy Erie’s lab at UNC and is now continuing her work in Caroline Kisker’s laboratory at the University of Wurzburg. Nucleotide excision repair involves multi-protein complexes, and she is working to isolate particular proteins to study their conformation with respect to each other and the DNA. These complexes are too small for their individual proteins to be located by AFM alone. The solution to this is to tag proteins with fluorescent dyes and collect both AFM and optical imagery of the specimen. This requires aligning the two images to a resolution much finer than that of the optical image.
Single-pixel registration of AFM and fluorescence was accomplished by repurposing a tool developed for the combination of AFM with Scanning Electron Microscopy (SEM) data, funded by the ARO and the ONR. This tool uses linear-least-squares registration of points tagged in the two modalities, in combination with projective texture mapping to display the fluorescence image in the AFM space and to enable direct manipulation with AFM, all while viewing and recording live fluorescence images. This tool was augmented during a recent trip by Resource graduate student Cory Quamman to the Kisker lab (travel and housing paid for by them). Cory added the FIONA kernel from our Spot Tracking project for subpixel alignment of fluorophores and a new maximum-locating kernel for AFM alignment to produce subpixel-accurate (for the fluorescence image) locations to feed to the least-squares solver. He calls this system Simple, Accurate Fluorescence and AFM Registration Imaging (SAFARI).