September 14th - Measuring and Manipulating Blood Flow in Individual Microvessels in Cortex: Nonlinear Optics Catalyzes New Ideas on Stroke

David Kleinfeld
Department of Physics
University of California, San Diego



I will review work on the dynamics of blood flow in the mid to upper layers of neocortex and show that flow is irregular at the level of capillaries. Further, the amplitude of stimulus-induced changes in flow is similar to that of the fluctuations, which suggests that changes in neuronal activation by natural stimuli does not compromise flow dynamics. I will next discuss spatial aspects of the fluctuations in flow, in which we perturb flow in individual vessels and measure the consequences of these perturbations in surrounding vessels. Our data show that flow in the superficial vascular network is rapidly rearranged to compensate for blockages in single vessels., while flow in deep capillary networks is seriously compromised by blockages. Novel tools for multi-site perturbations, including the use of high fluence ultrashort laser pulses, will be presented.





October 12th - Imaging Protein Kinase C in Living Cardiac Myocytes

Jeff Walker
Department of Physiology
University of Wisconsin-Madison



The heart is an electromechanical pump comprised of many cell types, the vast majority of which are rod-shaped ventricular myocytes. These cells express a wide variety of G-protein coupled receptors and protein kinases that regulate contraction strength, intracellular calcium, growth, apoptosis, resistance to hypoxia/ischemia, and the onset of heart failure. I will present our recent imaging studies of the function of G-protein coupled receptors and novel protein kinase C isoforms in cultured ventricular myocytes. The image shows a live rat ventricular myocyte 2 days after infection with adenovirus containing a GFP fusion protein of protein kinase C-epsilon. Treatment with a phorbol ester causes accumulation of protein kinase C-epsilon-GFP at the cell periphery and perinuclear region, and alters the cell's contractile response.





November 9th - Decoding Signals that Control the Actomyosin Response During Compensatory Endocytosis

Bill Bement
Department of Zoology
University of Wisconsin-Madison



During fertilization, Xenopus eggs undergo regulated, calcium-dependent exocytosis of cortical secretory granules. Within 15-20 s after exocytosis, Cdc42-dependent, actin “coats” assemble around the membranes of exocytosing cortical granules, and compress inward, thereby retrieving these compartments. Using 4D imaging in combination with a variety of molecular and pharmacological manipulations, we provide evidence that the formation of actin coats results at least in part from the recruitment of protein kinase Cb (PKCb) to exocytosing cortical granules, an event which is required for normal Cdc42 activation. PKCb recruitment, in turn, is mediated by two key upstream signaling events, which play complementary roles. The first event is calcium elevation itself, which rapidly recruits PKCb to all cortical granules (ie both those that have fused with the plasma membrane and those that have not). The second event is incorporation of diacylglycerol into those cortical granules that fuse with the plasma membrane, which results in selective retention of PKCb on exocytosing CG membranes. The rapid recruitment to all cortical granules is apparently controlled by the calcium- and phosphatidylserine-binding C2 domain of PKCb while the selective retention on exocytosing cortical granules is apparently controlled by the diacylglycerol-binding C1 domain of PKCb. These findings provide a molecular rationale for the observation that actin coats form only on those cortical granules that undergo exocytosis in spite of the fact that calcium is globally elevated.





December 14th - Multiphoton Microscopy and The Use of Nonlinear Excitation in Biology: Is it Useful in the Early Detection of Cancer?

Warren Zipfel
Department of BioMedical Engineering
Cornell University



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 8th - Functional MRI Studies of the Human Amygdala

Paul Whalen
Departments of Psychiatry and Psychology
The Waisman Laboratory for Brain Imaging and Behavior
University of Wisconsin-Madison



Recent studies of the human amygdala support decades of animal research implicating this brain system in threat assessment and aversive conditioning. Here we consider a broader role for the amygdala in vigilance associated with biologically-relevant predictive uncertainty. Human neuroimaging studies will be presented demonstrating differential responses across the amygdaloid region. Specifically, we aim to differentiate between regions of the amygdaloid complex that respond to stimuli providing a clear prediction of threat versus those that respond to stimuli that leave this prediction unclear. The implications of these results for the study of mood and anxiety disorders will also be considered.





March 8th - Novel Protein Labeling Technology for Cell Imaging

Georgyi Los
Promega Corporation
Madison, WI



The ability to specifically label proteins with markers that afford a wide range of optical properties and functionalities can help reveal information about protein function and dynamics in living cells. Here we describe a novel protein labeling technology developed for cell imaging, protein capture and immobilization applications. The technology is based on the efficient formation of a covalent bond between a specially designed reporting protein and its specific ligand, either in living cells, in solution, or on a solid support. The reporter protein is a genetically engineered, catalytically inactive derivative of hydrolase. The ligands are small chemical tags capable of carrying a variety of functionalities, such as fluorescent labels, environmental sensors, affinity handles, or attachments to a solid phase. The covalent bond forms rapidly under general physiological conditions, is highly specific, and essentially irreversible. The stability of the bond allows imaging of live cells during long periods of time, imaging of fixed cells, and multiplexing with different cell/protein analysis techniques. The open architecture of the technology ensures interchangeability of ligands, thereby facilitating a variety of functional studies (including imaging at different wavelengths and temporal or spatial separation of protein pools) without requiring changes to the underlying genetic construct.





April 12th - The Use of Multi-Photon Microscopy for Studies of the Structure and Function of the Kidney

Ken Dunn
Department of Medicine
Indiana University



Our group is actively involved in developing biological applications of multi-photon microscopy. In particular, we have developed methods of intravital multi-photon microscopy that allow us to analyze the physiology of the kidney of living rats and mice. We have also applied multi-photon microscopy to studies of kidney development, for which we have developed special analysis software that supports real-time 3 and 4-dimensional volume rendering on standard desktop computers.





May 10th - Imaging Synaptic Plasticity in the Brain

Karel Svoboda
Cold Spring Harbor Laboratory
Howard Hughes Medical Institute



The mammalian neocortex is a structure of immense complexity and its function changes in response to novel experience. In which synapses does the brain store information? What happens at these synapses? I will outline an experimental program based on lasers and microscopy that may help answer these profound questions.