September 14th - Single Molecule Nanoscale Rulers

Shimon Weiss
The Department of Chemistry and Biochemistry
University of California, Los Angeles



Single molecule methods have come of age and are now able to decipher individual molecular events in the test tube and in living cells. Single-pair FRET (spFRET) is used to follow structural changes, translocation and dynamic transitions of the enzyme RNA polymerase during initiation of transcription and promotor escape. Peptide-coated inorganic semiconductor nanocrystals (quantum dots - qdots) are used as fluorescent probes in cellular imaging. The peptides provide both solubilization and functionalization of the nanocrystals. Such qdots are targeted to cell-surface receptors of ex-vivo cultured cancerous HeLa cells with an exquisite specificity. Qdot targeting allows to follow the trafficking of individual proteins in live cells.





October 7th - Knights of the Periodic

Gelsomina De Stassio
Department of Physics
University of Wisconsin-Madison



The microscopic distribution of elements is tremendously useful to address diverse problems at the cutting edge of biological and medical research. These include:

1. Gadolinium Neutron Capture Therapy, a new approach to fight glioblastoma, the most lethal brain cancer, affecting 12,000 Americans per year.
2. The redox state of cells.
3. Transition metal accumulation in Transmissible Spongiform Encefalopathies.
4. Bacterial metabolism and biomineralization.

The instrument we, knights of the periodic table, use to investigate these fascinating fields is called Spectromicroscope for the Photoelectron Imaging of Nanostructures with X-rays (SPHINX), and uses x-rays to illuminate the sample surface and stimulate the emission of electrons by photoelectric effect.





November 4th - Live Cell Imaging of Glucose-Stimulated Insulin Secretion

Dave Piston
Department of Molecular Physiology and Biophysics
Vanderbilt University



We have applied several quantitative biophotonic tools to the study of dynamic signaling processes associated glucose-stimulated insulin secretion. Fluorescence recovery after photobleaching (FRAP) is used to measure intracellular mobility, and we have developed a fluorescence resonance energy transfer (FRET) approach to determine enzyme activity in living cells. By using multi-colored Green Fluorescent Protein (GFP) mutants fused to the glucose sensing enzyme glucokinase (GK), we have discovered that the location and activity of beta cell GK is acutely regulated by glucose through an autocrine response to insulin. Since small changes in beta cell GK activity have large effects on insulin secretion, this regulation plays a significant role in the control of blood glucose concentration.





December 2nd - Mechanisms of Chemotactic Cell Migration

Anna Huttenlocher
Departments of Pharmacology and Pediatrics
University of Wisconsin-Madison



Over the past decade multiphoton microscopy has proven to be valuable tool for biological research. Two-photon laser-scanning fluorescence microscopy has enabled fluorescence imaging with subcellular resolution at depths into living specimens not previously possible. Studies in thick tissue explants and in living animals are becoming more common, driven by investigations in neurobiology, developmental biology, pharmacology, and cancer research. Imaging of intrinsic cellular and tissue components such as collagen using second harmonic generation is also now being routinely used in biological research. The development of approaches utilizing multiphoton microscopy and nonlinear excitation specifically for studies in cancer biology and possibly even clinical detection hold promise, but depend on resolving a number of issues. These include the finding diagnostic criteria comparable to those obtained from routine histological imaging, and developing new ways to access to target organs and tissues in living animals. A broad overview of multiphoton microscopy will first be presented, followed by possible solutions to address the above issues specific to cancer research. These will include the use of nonlinear fluorescence and second harmonic imaging for diagnosis, studies of the progression of cancer and the use of strategies such as endoscopy and temporal focusing to achieve deeper imaging in living specimens.





February 3rd - Regulation of Breast Cell Behavior by Collagen Matrices: Molecular and Imaging Approaches

Patricia Keely
Departments of Pharmacology and Biomedical Engineering
University of Wisconsin-Madison



Cellular interactions with extracellular matrices are important for governing the behavior of cells, including normal epithelial differentiation and the metastasis of carcinoma cells. We have been particularly interested in understanding breast cell interactions with collagen-rich matrices. To this end, we have imaged focal adhesions within the context of complex 3D matrices, and made comparisons for matrices of different physical properties. Additionally, in collaboration with John White’s laboratory, we have recently used multiphoton and second harmonic approaches to image unstained collagen in live samples of mammary gland, comparing normal and tumor tissues. From this, a novel understanding of cell interactions with collagenous matrices is emerging, and will be discussed.





March 2nd - Imaging Embryos in The Fourth Dimension

Jeff Hardin
Department of Zoology
University of Wisconsin-Madison



Tremendous advances in imaging within cultured cells have revolutionized cell biology at the beginning of the 21st century. In particular, analyzing the dynamics of GFP-tagged cell adhesion and cytoskeletal proteins has revealed dynamic aspects of cell adhesion that were previously impossible. Although similar cell adhesion events take place during embryonic development, gaining comparable glimpses of these processes in living embryos is challenging because they are thick, far from flat, and their overall shape is constantly changing. We and our colleagues have developed techniques and software to analyze four dimensional datasets that have allowed us to analyze the redistribution of cell adhesion proteins during embryonic development in C. elegans embryos. These technologies have provided some of the first glimpses into the dynamics of cell adhesion in living embryos, and how specific proteins regulate these dynamics.





April 6th - Dynamic Imaging of Protein Molecules: FLIM-FRET Microscopy

Ammasi Periasamy
W.M. Keck Center for Cellular Imaging
University of Virginia



In this study, we used two-photon excitation FRET-FLIM microscopy techniques to visualize the interactions of the transcription factor CAATT/enhancer binding protein alpha (C/EBPa) in living pituitary cells. We also demonstrate interaction between the proapoptotic protein BAD and the prosurvival protein Bcl-xL within traumatic axonal injury (TAI) following traumatic brain injury (TBI) [5]. Biorad Radiance2100 confocal/multiphoton system was configured to integrate the Becker-Hickl TCSPC lifetime imaging board to acquire the FRET-FLIM images.





May 10th - Monitoring Naturally Occurring Synapse Elimination in Fluorescent Neonatal Mice

Jeff Lichtman
Department of Molecular and Cellular Biology
Harvard University



My colleagues and I use transgenic mice that express fluorescent proteins in neurons to monitor the remodeling of synaptic circuits that takes place in the developing nervous system. This remodeling plays a critical part in the way young animals use experience to mold their nervous systems to conform to the world they live in. Our work focuses on the synaptic connection between motor neurons and muscle fibers. In adults, each muscle fiber is innervated by exactly one motor neuron and at just one site, the neuromuscular junction. Each motor neuron however distributes its innervation to a number of muscle fibers. This pattern emerges in early postnatal life as synapses of different motor neurons that initially multiply innervate neuromuscular junctions are eliminated. Time-lapse imaging in vivo of muscles in which different axons express different colored fluorescent proteins reveals the way neuromuscular junctions undergo the transition from multiple to single innervation and motor axon branches are removed. Two-photon laser axotomy shows that the elimination is driven by competition between axons that transiently co-occupy synaptic sites. Serial TEM implicates a role for glia in the removal process.

References: Feng et al. Neuron 2000; 28:41-51; Keller-Peck, et al. Neuron 2001; 31:381-94; Walsh and Lichtman Neuron, 2003, 37:67-73; Kasthuri and Lichtman Nature, 2003, 424:426-430; Buffelli et al. Nature, 2003, 424:430-434 .