Three-dimensional localization of immunogold markers using two tilted electron microscope recordings.

A method is presented to determine the three-dimensional positions of immuno-labeled gold markers from tilted electron micrograph recordings by using image processing techniques. The method consists of three basic modules: localization of the markers in the recordings, estimation of the motion parameters, and matching corresponding markers between the views. Localization consists of a segmentation step based on edge detection and region growing. It also allows for the separation of (visually) aggregated markers. Initial estimates for the motion parameters are obtained from a small number of user-indicated correspondences. A matching algorithm based on simulated annealing is used to find corresponding markers. With the resulting mapping, the motion parameters are updated and used in a new matching step, etc. Once the parameters are stable, the marker depths are retrieved. The developed method has been applied to semithin resin sections of A431 cells labeled for DNA and detected by silver-enhanced ultrasmall gold particles. It represents a reliable method to analyze the three-dimensional distribution of gold markers in electron microscope samples.

Structural hierarchy in the clustering of HLA class I molecules in the plasma membrane of human lymphoblastoid cells.

Major histocompatibility complex (MHC) class I antigens in the plasma membranes of human T (HUT-102B2) and B (JY) lymphoma cells were probed by immunochemical reagents using fluorescence, transmission electron, and scanning force microscopies. Fluorescent labels were attached to monoclonal antibodies W6/32 or KE-2 directed against the heavy chain of HLA class I (A, B, C) and L368 or HB28 against the beta 2-microglobulin light chain. The topological distribution in the nanometer range was studied by photobleaching fluorescence resonance energy transfer (pbFRET) on single cells. A nonrandom codistribution pattern of MHC class I molecules was observed over distances of 2-10 nm. A second, nonrandom, and larger-scale topological organization of the MHC class I antigens was detected by indirect immunogold labeling and imaging by transmission electron microscopy (TEM) and scanning force microscopy (SFM). Although some differences in antigen distribution between the B- and T-cell lines were detected by pbFRET, both cell lines exhibited similar clustering patterns by TEM and SFM. Such defined molecular distributions on the surfaces of cells of the immune system may reflect an underlying specialization of membrane lipid domains and fulfill important functional roles in cell-cell contacts and signal transduction.

The scanning force microscopy of DNA in air and in n-propanol using new spreading agents.

We present recent advances in DNA specimen preparation technique for scanning force microscopy (SFM) based on spreading on mica in the presence of cationic and non-ionic detergents. Reproducible DNA imaging in air and in n-propanol has been achieved in the presence of the non-ionic detergent 2,4,6-tris(dimethylaminomethyl) phenol (DMP-30) or the cationic detergent cetylpyridinium chloride (CP) in a microdrop containing nanograms of DNA. In an alternative procedure, a microdrop of detergent is applied to the surface just prior to the DNA. Quantitative image analysis yields as the apparent molecular dimensions of the DNA a width of approximately 7 nm and a height of approximately 0.7 nm, and delineates the problems of DNA metrology by SFM.