September 10th - Extracting More Dimensions of Data from Fluorescence Images

John White
Laboratory for Optical and Computational Instrumentation (LOCI)
University of Wisconsin-Madison

Conventional fluorescence microscopy yields simple intensity images sampled at 1 to 3 wavelength bands. However, there is considerably more information available in a fluorescence image that can contain useful data. Fluorescent molecules exhibit a characteristic lifetime that is also dependent on the microenvironment of the molecule. This information can serve to identify the molecule or report on the molecule's environment. Fluorescent molecules also exhibit a characteristic emission spectrum that can provide more information concerning the identity of the molecule than two or three images taken at different spectral bands. Microscopes are being developed that can measure and display lifetime and fluorescence spectrum at each pixel of an image. Such instruments should allow better discrimination of multiple fluorophores with overlapping spectra and will facilitate FRET measurements.

October 8th - Noninvasive Imaging of Temperature and Thermal Lesion Formation in-vivo During Radio Frequency Ablation

Tomy Varghese
Department of Medical Physics
University of Wisconsin-Madison

Hepatocellular carcinoma (HCC) is one of the most common solid organ malignancies worldwide, with an annual incidence of at least 1 million new patients. Optimal therapy of both primary and metastatic liver disease is complete surgical resection with negative margins. However, most (80-90%) HCC patients are not candidates for surgical resection because of tumor size, location, or hepatic dysfunction related to cirrhosis. The high post-operative complication rate coupled with the low number of resection candidates has increased the interest in minimally invasive treatment, such as focal ablative therapies for both primary and secondary liver tumors. Radio frequency ablation (RFA) is an interstitial focal ablative therapy that can be used in a percutaneous fashion and permits in situ destruction of hepatic tumors. However, local recurrence rates after RFA therapy are as high as 34-55%, due in part to the inability to accurately monitor temperature profiles in the tissue being ablated, and to visualize the subsequent zone of necrosis (thermal lesion), leading to the incomplete ablation of the tumor, generally areas near the tumor edges.

Methods for real-time monitoring of the spatial distribution of heating to better control the degree of tissue damage, in addition to elastographic visualization of the thermal lesions will be presented. Temperature monitoring is currently performed using thermosensors on the tines of the RFA probe. Temperature estimates obtained using a cross-correlation algorithm applied to RF ultrasound echo signal data acquired at discrete intervals are used to continuously update a thermal map of the treated region during RFA.

November 12th - Calcium Signals in Non-Excitable Cells: Specific Signals from Generic Building Blocks

David Yule
Department of Pharmacology and Physiology
University of Rochester Medical Center

Changes in cytosolic calcium represent a ubiquitous system in biology controlling countless cellular processes. Our research focuses on gaining insight into the mechanisms which give rise to the specificity and fidelity of calcium signaling events in non-excitable cells. Our working hypothesis is that the specific kinetics and the sub-cellular localization of calcium signals encode information, important for the activation of distinct physiological endpoints. In testing these ideas we utilize approaches such as confocal microscopy to evaluate the localization and numbers of particular calcium signaling “modules” and then relate this information to the characteristics of calcium signals that are evoked. To monitor calcium signals with high temporal and spatial resolution we utilize contemporary biophysical techniques such as time-resolved fluorescence imaging, electrophysiology and flash photolysis of caged compounds. In particular we are interested in correlating the specific characteristics of calcium signals to cellular processes such as gene transcription or activation of ion channels.

December 10th - Development of Molecule-Targeted Contrast Media for Molecular Imaging: Correlative MRI, EEL, and EDX Spectroscopy

Marek Malecki
Department of Animal Sciences
University of Wisconsin-Madison

With the rapid advances in functional genomics and proteomics, there is an urgent need for structural and functional information on how the myriad gene products of the cell combine and interact to form biomolecular assemblies, biomolecular "machines'' organelles, and cells. Moreover, dramatic progress in molecular medicine allows us to streamline that information into the interpretation of mechanisms of diseases. These rapidly growing areas of research and medicine call for molecular imaging technology that would allow us to pinpoint the location of many individual molecules within complex biomolecular assemblies, and within the entire human body in health and pathology. Such an imaging technology does not exist at this time. In an attempt to come up with a molecular imaging strategy, we promote correlative imaging approaches in which nuclear magnetic resonance imaging (MRI) works in concert with energy dispersive x-ray spectroscopy (EDS), and electron energy loss spectroscopy (EELS). Ideally, the same markers are detectable with various imaging techniques. Moreover, they are detectable with other research instruments, allowing us direct interdisciplinary correlation of the data. One of the routes relies upon engineering of markers which would target selected molecules - targeted contrast agents, while carrying reporter molecules detectable with MRI, EELS, and EDS. The antibody-based markers should facilitate correlations of the data on gene expression available from biochemistry, molecular biology, and molecular imaging laboratories, as well as the location and functions of the gene products. In this sense, molecular imaging serves as an integrative factor for life science endeavors.

February 4th - Computational Approaches to Multi-Dimensional Imaging

Kevin Eliceiri
Laboratory for Optical and Computational Instrumentation (LOCI)
University of Wisconsin-Madison

Relatively recent strides in non-invasive medical imaging and in vital microscopy have yielded a new form of massive data type - the multichannel, three-dimensional, timecourse image recording. These multidimensional data sets document the dynamic changes within the full volume of a specimen over time, often simultaneously monitoring several different parameters. Examples include: i) microscope image recordings of the distinct signals produced by a reporter gene product and an organelle-specific chemical probe during the development of an embryo; ii) MRI tomography of a live patient's tissues responding to a treatment; iii) a reconstruction of serially sectioned fixed specimens representing sequential stages in the changing ultrastructure of a particular cell type. While the imaging modalities used to generate the data differ greatly, they all share the common attributes of being multidimensional and are usually represented by fairly large data archives. To help meet the computational challenge of the effective analysis of this data we are developing a framework for the advanced analysis and manipulation of multidimensional data. With the software tools of this framework, these massive data archives could be more readily explored to extract all possible information and detail present in each dimension.

March 4th - MR Tracking of Magnetically Labeled Cells After Transplantation

Jeff Bulte
Department of Radiology
John Hopkins University

Stem cells have the potential to treat and possibly cure a variety of disorders, particularly those of the central nervous system. To develop successful clinical therapies, the fate of these cells after grafting must be monitored in a noninvasive manner. By incorporating magnetic nanoparticles into (neural stem cell-derived) oligodendrocyte progenitor cells, we have developed methods that enable their detection using MRI. Following grafting of cells in the spinal cord of 1 wk old myelin-deficient rats, progenitor cell migration within the spinal cord, mainly along the dorsal column, could be easily identified by high resolution MR imaging ex vivo. We also observed an excellent agreement between the areas of MR contrast enhancement and immunohistochemical stainings for newly formed myelin. Furthermore, when transplanted in the ventricles of Shaker rats, we found that tagged cells could be readily detected in vivo at least up until 6 weeks following transplantation, with a good correlation between the obtained MR contrast and staining for b-galactosidase expression (a traditional marker gene). We conclude that, following transplantation, magnetically labeled cells can be accurately and repeatedy monitored in vivo; this technique has the potential to be implemented in a clinical setting in order to help guide stem cell-based therapies. Cell transplantation studies in other disease models are currently in progress, including spinal cord traumatic injury, experimental autoimmune encephalomyelitis, viral-induced lower motor neuron disease, and Parkinson's disease.

April 1st - Optical Spectroscopy and Imaging of Neoplasia

Nirmala Ramanujam
Department of Biomedical Engineering
University of Wisconsin-Madison

Fluorescence imaging has emerged as a promising technique for the early detection of pre-cancer and cancer, a capability that is critical for more effective treatment of this disease, and improved survival rates. Fluorescence imaging is achieved by exciting fluorophores in an area of tissue with specific wavelength(s) of light and measuring the fluorescence response, thus extracting information about the concentration, location, and environment of the fluorophores. The sensitivity and resolution of this technology depends on the ability to establish a source of contrast between the neoplastic and non-neoplastic tissue. This contrast may come from preferential or exclusive presence of the fluorophore within the cancerous tissue, as with exogenous fluorophores, or it may come from more subtle changes in the optical and metabolic characteristics of diseased versus non-diseased tissue, as with endogenous fluorophores. In either case, it is desirable to identify techniques that maximize contrast, and improve resolution and sensitivity. This presentation will review fluorescence based techniques for imaging neoplasia in vivo. Progress in instrumentation, algorithms and contrast agents will be discussed. Applications that will be presented include pre-cancer and cancer detection in human studies and molecular imaging in living animal models.

May 6th - Using Two-Photon Time Lapse Microscopy to Study Learning in Cultured Networks

Steve Potter
Coulter Department of Biomedical Engineering
Georgia Institute of Technology

The Laboratory for Neuroengineering at Georgia Tech applies engineering and neurobiology approaches to study basic neural mechanisms and apply them to engineered systems. As one of six groups in the Laboratory, the Potter Group uses networks of cultured mammalian cortical cells as a model system to study learning, memory, and information processing in neuronal networks. We grow dissociated cultures on multi-electrode arrays (MEAs) for up to two years, and use these to form a two-way communication link between the network and a computer. With this scheme, we can allow the network to express its activity as behavior in a simulated animal (animat). The hybrid of a living network and a robotic body we call a 'Hybrot'. We are devising mappings between neural activity and behavior, and between sensory input and stimulation, that will enable the hybrot to learn and adapt to its environment. We will have the unprecedented opportunity to observe the learning process at the cellular (and subcellular) level, because the 'brain' is growing on a flat glass substrate where we can image every cell in the network. Two-photon microscopy is crucial for carrying out repeated imaging of the morphology of neurons and glia as they participate in functional plasticity. With this technique we can observe sub-micron changes, for example, in the structure of dendritic spines, as a result of simultaneously recorded electrical activity and stimulation. We employ genetically labeled neurons from transgenic mice expressing various fluorescent proteins, to further extend the time scale of observation compared to using fluorescent dyes. Relevant publications can be found here.