766-Pos Board B566 Fluorescence Correlation Spectroscopy of Tryptophan-Containing Proteins in Sugar Solutions Nathan Holman, Yuli Wang, David Sidebottom. Cryopreservation has become an important avenue of molecular and cellular biology research in recent decades but the mechanism by which even simple cryopreserving agents such as sugar solutions protect biomaterial remains poorly understood. To investigate the behavior of proteins in sugar solutions, we have undertaken a study of sugar solutions using Photon Correlation Spec- troscopy (PCS) that reveals a strong tendency for sugars to cluster even at dilute concentrations. To investigate tryptophan-containing proteins dissolved in these solutions, we have also implemented Fluorescence Correlation Spectros- copy (FCS) using a two-photon excitation approach as a selective probe of protein dynamics. 767-Pos Board B567 Reconstruction of Nanostructures in Cells by the Modulation Tracking Optical Method Enrico Gratton, Michelle A. Digman, Luca Lanzano’. Resolving biological structures at the nanoscale has been a subject of intense recent research. Essentially two approaches have been proposed, one based on shaping the effective illumination profiles using stimulated emission (STED) and the other using the determination of the center of mass emission by single molecules (PALM and STORM). Both approaches have been shown to pro- duce high quality images with resolution on the order of 20 nm. The STED method is ultimately based on using a raster scan approach to produce an im- age, which is quite inefficient for imaging objects in 3D and the PALM method is very slow for obtaining 3D structures of the size of microns. We have developed the modulation tracking method which is based on a feedback principle that is capable of producing 3D images with resolution in the 20 nm range. With simulations of 3D objects we show how the images are acquired in 3D and projected in 2D screen and we determine the size limits in imaging very small object. We also show that the modulation tracking method pro- duces images based on the location of the surface of objects with high accu- racy and we compare the images obtained by the modulation tracking approach with the 3D reconstruction methods based on the determination of intensity iso-surfaces. 768-Pos Board B568 Advances in Three Dimensional Super Resolution Fluorescence Localiza- tion Microscopy Michael J. Mlodzianoski, Joerg Bewersdorf. For centuries, the resolution of light microscopes has been restricted by the dif- fraction limit of light, typically ~250nm. Over the last two decades several methods have been developed to get around these limitations. In particular, FPALM, PALM, and STORM, utilize stochastic optical switching of photoac- tivatable fluorescent molecules and their sequential localization to generate su- per-resolution images. After the first generation, which only offered lateral (two dimensional) improvements to resolution, recent developments now achieve 3D super-resolution. The challenge of enhancing resolution in the axial (depth) direction requires breaking the symmetry of the point spread function around the focal plane. Here, we focus on Biplane Fluorescent Photoactivatable Local- ization Microscopy (BP-FPALM) which utilizes multiplane detection to local- ize particles with sub-100 nm precision in 3D. BP-FPALM has been previously described and characterized. Here we demon- strate two color imaging of the mitochondrial matrix and mitochondrial nucleoids with a variety of photoactivatable probes. In addition, we show BP-FPALM’s capability of measuring samples of a thickness greater than 5 mm while also containing a large number of localized particles. For image ac- quisition times longer than a few seconds, sample drift can blur images and re- duce resolution. We introduce algorithms to correct and account for any drift. 769-Pos Board B569 Synthesis of Dopamine in Pancreatic b-Cells and Its Impact on Glucose Stimulated Insulin Secretion Alessandro Ustione, David W. Piston. Glucose homeostasis is maintained by small clusters of hormone secreting cells in the pancreas: the pancreatic islets. Insulin secreting beta-cells make up ~ 90% of each islet and secrete insulin in a tightly regulated manner. Understanding the mechanisms that regulate insulin secretion is a key factor in developing therapies for type-2 diabetes and metabolic syndrome. Evidence shows that dopamine inhibits glucose stimulated insulin secretion (GSIS) in vitro, and the effect is mediated by the D2 isoform of the dopamine receptor. Yet, there is no evidence of dopaminergic neurons innervating pan- creatic islets, and therefore, the biological relevance of such sensitivity is not clear. We demonstrate that pancreatic islets can produce dopamine from the circulat- ing precursor L-dopa and that the resulting dopamine is released as an autocrine inhibitory signal to regulate insulin secretion. We generated data from wild type and transgenic mice lacking D2 dopamine receptor or the dopamine transporter (DAT) to understand how dopamine is producing its effects. We monitor islet metabolic activity by imaging of NAD(P)H autofluorescence with two photon excitation and we measure intracellular [Ca 2þ ] i oscillations by confocal microscopy. We show that dopamine does not affect b-cell meta- bolic activity. Instead dopamine and L-dopa reduce the frequency of [Ca 2þ ] i oscillations and this correlates with reduced glucose stimulated insulin secre- tion (GSIS). This suggests that dopamine receptor activation affects GSIS downstream of glucose metabolism, probably reducing Ca 2þ influx through the plasma membrane. This finding provides a new target for drug development to treat type-2 diabetes, and it may explain reported side effects of second generation antipsy- chotics that result in elevated risk of type-2 diabetes and impaired glucose tolerance. 770-Pos Board B570 Direct Observation of Single Oligomers of the Alzheimer’s Amyloid-b Peptide on Live Cell Membranes Robin Johnson, Joseph Schauerte, Kathleen Wisser, Indu Saluja Igo, Ari Gafni, Duncan Steel. Oligomeric species of the amyloid-b peptide are strongly implicated in the synaptic dysfunction and neuronal loss seen in Alzheimer’s disease. While proposed mechanisms for oligomeric toxicity are abundant and diverse, many involve amyloid-b (Ab) interaction with cell membranes, either via di- rect insertion into the cell membrane or by binding to specific cell-surface receptors. One such hypothesis holds that Ab forms calcium-permeable chan- nels within the membranes of neurons, disrupting homeostasis and triggering an apoptotic signaling cascade. However, both the exact identity of the toxic aggregate or aggregates and the mechanism by which the toxicity is mediated remain undetermined. Study of this system under physiological conditions presents a challenge, as oligomeric forms of the peptide in vivo are heteroge- neous and metastable, and Ab itself is normally present in the brain at only nanomolar concentrations. We recently used single molecule spectroscopy to detect stable, conductive low-order Ab(1-40) oligomers in synthetic mem- branes exposed to nanomolar levels of the peptide. Similar oligomers may form on the membranes of live cells and, by disrupting cell membrane integ- rity, contribute to the functional abnormalities and neuronal death observed in Alzheimer’s. With single molecule confocal laser scanning microscopy, we have identified small Ab(1-40) oligomers bound to the membranes of SH-SY5Y neuroblastoma cells after ten minutes’ exposure to low (50nM) peptide concentrations. Additionally, we have used single-particle fluores- cence intensity measurements to characterize the oligomeric states of these cell-bound aggregates. We find that small oligomers ranging from dimers to octamers form in solution under physiological conditions and that the size distribution shifts towards larger oligomers for Ab(1-40) on the cell membrane, which may indicate that oligomer growth can occur following membrane binding. 771-Pos Board B571 Macromolecular Crowding and Stem Cell Differentiation Rafi Rashid, Michael Raghunath, Thorsten Wohland. Macromolecular crowding (MMC) is a biophysical tool which has been used extensively to enhance chemical reactions and biological processes by means of the excluded volume effect (EVE). The in vivo stem cell microenviron- ment contains macromolecules which are crucial for stem cell self-renewal and cell fate determination. In order to mimic this physiological microenvi- ronment, crowders are included in cell culture medium. We have observed that the ex vivo differentiation of human mesenchymal stem cells (hMSCs) into the adipogenic lineage is significantly amplified when a crowder mixture comprising Ficoll 70 and Ficoll 400 is added to the culture medium. Stem cell differentiation is modulated by soluble chemical substances as well as interactions between cells and the extracellular matrix (ECM), and both these external influences may be affected by MMC. Measurements we have per- formed by fluorescence correlation spectroscopy (FCS) on bulk solutions in- dicate that crowders change diffusion coefficients via changes in viscosity. Our data do not show that Ficoll additives cause anomalous subdiffusion within a crowder concentration range of 0 to 300 mg/ml. The diffusion of fluorophore-labelled molecules in artificial lipid bilayers and membranes of living cells is not changed by crowders, suggesting that these crowders do not directly alter membrane properties and cell surface signalling. However, we have data to suggest that crowders exert an effect on biomolecular inter- action kinetics. We have also observed that crowders are taken up by stem 142a Sunday, March 6, 2011