S phase accumulation. Redox changes with tocopherol or ferricyanide or in- creases of the ATP energetic pool with pyruvate will suppress autophagy allow- ing for DNA and cell repairs and completion of cell cycles for cell proliferation. This process of unlocking the cell cycle with EMF to optimizing the effect of the topical treatment has previously been shown increase drug chemotoxicity (taxol, DiHydroArtemisinin, .) in tumor cell lines. 3653-Pos Board B514 Microwave Sensor for Measuring the Growth of Biomaterials Christopher E. Bassey, Sarah A. Mintah, Mary C. Bassey. Azusa Pacific University, Azusa, CA, USA. The propagation of electromagnetic waves in a transmission line is affected by the dielectric permittivity and thickness of the material surrounding the line. We present a technique that can determine the growth of biomaterials from measurements of wave propagation characteristics. It utilizes a coplanar trans- mission line as sensor and an Agilent Automatic Network Analyzer for mea- surements in the time domain. The test material is placed on one side of the sensor. Numerical analysis indicates that the phase velocity, effective dielectric constant, and characteristic impedance show dependence on the thickness of the test dielectric. Experimental verification was obtained using water as the test dielectric. This technique can be used for continuous monitoring of the growth or thickness of biomaterials. Imaging & Optical Microscopy: Superresolution Imaging & Single Molecules 3654-Pos Board B515 Simultaneous Observation of the Three-Dimensional Orientation and Position of a Single Fluorescent Probe Masashi Ohmachi 1 , Yasunori Komori 2 , Atsuko H. Iwane 1 , Fumihiko Fujii 1 , Takashi Jin 1,3 , Toshio Yanagida 1,3 . 1 Osaka University, Osaka, Japan, 2 University of Tokyo, Tokyo, Japan, 3 RIKEN, Osaka, Japan. We have developed a new microscopy system for simultaneously measuring the three-dimensional orientation and position of a fluorescent probe based on the principal of polarization analysis. This method requires only that one collect fluorescence counts from a single fluorescent probe at four different po- larizations such that only relatively simple optics and mathematical equations are required to yield the orientation. Orientations can be determined with less than 10  accuracy at 33ms time resolution except for a situation where a probe is nearly parallel to optical axis. We used the newly developed microscopy and quantum rods as our fluorescent probes to simultaneously measure myosin V movement along an actin filament and rotation around its own axis. Myosin V was seen to rotate 90  around its own axis for each step. From this result, we suggest that in the two-headed bound state the necks are perpendicular to one another, while in the one-headed bound state the detached trailing head is biased forward in part by rotating its lever arm about its own axis. This new microscopy system should have particular applicability to biophysical studies that investigate single molecule orientation dynamics. 3655-Pos Board B516 Accurate Quantification Methods for Single Molecule Localization Avtar Singh, Han Gao, Alexander Song, Rebecca Williams, Warren R. Zipfel. Cornell University, Ithaca, NY, USA. Super-resolution and single molecule imaging methods often rely on photon counts for quantification of precision and image processing to improve particle detection and localization. However, there is still no consensus on an accurate photoelectron calibration method for EMCCDs. In order to directly measure the light response of an EMCCD, we illuminate each sensor individually with highly attenuated laser light and compare pixel values to readout from a cali- brated photomultiplier tube in photon counting mode. The data is then com- pared to manufacturer specification sheets as well as various published calibration methods and results from EMCCD theory. Meanwhile, smoothing and background subtraction are often used to improve the quality of single mol- ecule data, but the effects of such routines on localization and spot-finding have not been rigorously studied. Furthermore, precision is usually calculated based on a simple model of nonlinear least-squares Gaussian fitting of the point spread function in which the effects of denoising are not at all considered. We apply several methods for image filtering and background subtraction to synthetic images representative of PALM/STORM data and measure their ef- fects on localization precision, bias and spot-finding (quantified using Type I and Type II error rates). 3656-Pos Board B517 Fluorescence Excitation and Imaging of Single Molecules Near Coated Surfaces: A Theoretical Study Daniel Axelrod. University of Michigan, Ann Arbor, MI, USA. Microscopic fluorescent samples of interest to cell and molecular biology are almost always in aqueous medium near a solid surface. Frequently, that surface is coated with a thin film such as: a lipid monolayer, bilayer, or multilayer; a col- lagen or agarose layer deposited before cell plating or deposited by the cells themselves; acrylamide gel to immobilize beads or single molecules (such as in commercial preparations for nucleic acid sequencing); or a cell wall inter- posed between the substrate and cellular organelle. Both excitation and emis- sion of fluorescent single molecules near film-coated surfaces are strongly affected by the proximity of the coated surface, the film thickness, its refractive index, and the fluorophore’s orientation. For TIR excitation, multiple reflec- tions in the film lead to unique resonance peaks in the evanescent intensity vs. incidence angle curve. For emission, multiple reflections coupled to the flu- orophore’s near field emission create a distinct intensity pattern in the back fo- cal plane (BFP) of a high aperture objective. In principle, these features should allow retrieval of information about local film thickness and refractive index, and about fluorophore axial position and 3D molecular orientation. This en- tirely theoretical analysis with computer-generated images explores these pos- sibilities. Supported by NIH grants 2R56NS038129-11 and 1R21NS073686-01 to DA and Ronald V. Holz. 3657-Pos Board B518 Super-Resolution Imaging of Membrane Topology using Small Molecule Lipophilic Dye Tulip Nuwal, Wei Zhang, Bo Huang. University of California at San Francisco, San Francisco, CA, USA. Plasma membrane of any cell supports a vast array of biomolecules on its sur- face. These are crucial in response and adaptation to the external environmental cues of the cell. The process of determining these cues is done by a plethora of plasma membrane associated proteins including receptors, channels and pores. The spatial organization including compartmentalization of these proteins is critical for cellular function. In order to generate a high spatial resolution topo- logical map of a plasma membrane we use wide-field fluorescence super- resolution imaging technique known as Stochastic optical reconstruction microscopy (STORM). STORM works on the principle of stochastic switching of single-molecule fluorophores and precise localization of the centers of these spots. Here we labeled the plasma membrane using a small molecule lipophilic carbocyanine dye, DiD, which can photoswitch under similar conditions as pre- viously used for Cy5 photoswitching. We stained Drosophila Schneider 2 cells with DiD and imaged them under both fixed and live conditions using 3D STORM imaging and/or single-particle tracking modes. We observed plasma membrane features including clathrin coated pit-like structures. Further exper- iments using multicolor STORM to image DiD together with membrane pro- teins will shed light on how membrane protein distribution is correlated with membrane topology. 3658-Pos Board B519 Three Dimensional Single Molecule Localization using Phase Retrieved Pupil Functions Sheng Liu, Keith A. Lidke. University of New Mexico, Albuquerque, NM, USA. Single particle tracking and single-molecule localization based super- resolution techniques rely on the precise and accurate localization of single fluorescent molecules. For two dimensional imaging, relatively simple models of a microscope point spread function (PSF), such as the two dimensional Gaussian, are adequate for most common imaging approaches. However, three dimensional localization is hampered by the fact that the image of a molecule near the focal plane contains little information about its axial position. In recent years, several three dimensional (3D) imaging techniques have been demon- strated that improve the localization of single fluorescent molecules along the axial direction, such as astigmatic imaging [1] , a double helix point spread function (DH-PSF) [2] , and dual focal plane methods [3] . Although these methods implement very different optical setups, they use either a simple Gaussian models, theoretical PSFs that do not account imaging system aberrations, or large, unwieldy, experimentally acquired 3D PSFs that can be prone to arti- facts. Here we introduce a localization algorithm based on phase retrieved pupil functions. Pupil functions can contain information about specific aberrations present in the imaging system and can be used to calculate realistic 3D PSFs from a small set of Zernike polynomial coefficients that describe the pupil mag- nitude and phase. This compact representation of the 3D PSF allows the PSF to be efficiently calculated as needed in an iterative update method implemented Wednesday, February 29, 2012 721a