Quantitative Imaging

Electron Microscopy

UNM investigators Wilson and Oliver have a long history of using scanning and transmission electron microscopy to study cellular features that influence signal transduction. The use of immunogold-labeled whole cells and membrane sheets to study membrane topography is a unique strength of the group. The SEM image at top right, showing the distribution of 15 nm gold-labeled IgE receptors on the outer surface of an intact activated mast cells illustrates the preferential clustering of receptors away from membrane ruffles. The TEM image at lower right, showing an unusual ring of PI 3-kinase (5 nm gold) around activated ErbB3 receptors (10 nm gold) on the cytoplasmic face of a breast cancer cell membrane, demonstrates the power of the membrane sheet technique to visualize previously unknown spatial relationships between receptors and their signaling partners.

Reconstruction of 3-D cell structure is also an emphasis of the electron microscopists, with the specific goal of providing mathematical modeling partners with accurate geometry for simulating signaling in a cellular context. The image at right shows the spatial relationships of the endoplasmic reticulum (yellow), mitochondria (blue) and plasma membrane (red) in RBL-2H3 cells. It was reconstructed from an electron tomographic tilt series (performed at the National Center for Microscopy and Imaging Research, UCSD).

Fluorescence-based Technologies

Fluorescence-based Technologies to Quantify Diffusion, Protein-Protein Interactions

The combined expertise of UNM investigators Diane and Keith Lidke in biophysics and bioimaging keeps the STMC ahead of the technology curve in quantitative fluorescence microscopy microscopy. Intensive effort has been invested in the use of multi-color quantum dot probes for imaging the movements of cell surface receptors, as well as capturing such processes as receptor dimerization and internalization in real time. The image at left, taken from Andrews et al., Nature Cell Biology 2007, demonstrates the use of monovalent IgE-quantum dot probes to visualize the trapping of an individual IgE receptor within a dynamically-rearraging network of actin "corrals.” The repertoire of available fluorescence microscopy methods also includes TIRF (Total Internal Reflection), FRAP (fluorescence recovery after photobleaching) and FRET (Förster resonance energy transfer). Under development are these exciting new technologies: STORM (with J. Timlin at SNL) and SML-SR (Single Molecule Localization based Super-Resolution), using photo-switchable dyes.

The concept of Single Molecule Localization based Super-Resolution (SML-SR).

Left: Probes that have intermittent fluorescence can be imaged individually. Right: super-resolution images can be built by placing Gaussian profiles with width sloc at the found position. Bottom left: Conventional image, Bottom right: Reconstructed super-resolution image. Scale bar: 100 nm.

Image Analysis

Led by S. Steinberg, the UNM image analysis team has developed sophisticated image processing and spatial statistics applications to acquire quantitative information from electron microscopy images, providing rich data sets for spatial stochastic models.

A complementary team led by K. Lidke is generating novel algorithms and software for the analysis of single particle tracking data generated by confocal, TIRF and hyperspectral microscopy.