Fluorescent Proteins for Super-Resolution Microscopy

Tuesday, February 14, 2017 fluorophores these two effects can be distinguished, and the oligomerization state of a labeled protein of interest can be estimated. We therefore show how the theoretical framework developed for membrane proteins needs to be adjusted to account for this additional degree of freedom in soluble proteins. Overall, bulk homo-FRET and laser photobleaching is a promising method to determine the oligomerization state of a protein of interest, which can have a low concentration (0.1-0.5 mM) and needs only a single fluorescent label. The method requires only a photometer or microplate reader capable of measuring steady-state anisotropy. 2226-Pos Board B546 Optimizing a Time-Resolved Spectrometer for All Time Scales Christian Litwinski, Sebastian Tannert, Manoel Veiga, Felix Koberling, Marcus Sackrow, Michael Wahl, Olaf Schulz, Marcelle Koenig, Rainer Erdmann. PicoQuant GmbH, Berlin, Germany. Time-resolved fluorescence spectroscopy is a spectroscopist’s most valuable tool for the investigation of excited state dynamics in molecules, complexes, or semi-conductors. In recent years, the study of luminescence properties has gained in popularity in many scientific fields, including Chemistry, Biology, Physics, as well as in Life, Material or Environmental Sciences. The investigations to be carried out in each of these fields impose different requirements. On one side, monitoring dynamic processes in the excited state necessitates high time resolution that can be achieved by fast pulsed lasers and detectors along with appropriate time-correlated single photon counting (TCSPC) units and small monochromators. On the other hand, high spectral resolution is desirable for fluorophore characterization, requiring detectors with high quantum efficiencies, flash lamps for phosphorescence measurements and large monochromators. Up to now, spectrometers have been usually targeted towards either one of these two specifications. Spectrometers equipped with hybrid detectors, versatile TCSPC cards with optional longer time ranges, and pulsed lasers capable of working in a burst mode can offer an combined solution, covering most of the demands of either high time or spectral resolution. We will demonstrate the performance of such a spectrometer in terms of its time resolution, the ability to measure long decays and record time-gated spectra using laser drivers with burst capabilities. This type of instrument is of great value for analytical facilities in research centers, as it offers a wide range of possible spectroscopic applications in a single, easy to use instrument. 2227-Pos Board B547 Hyperspectral Measurements Allow Separation of FRET Signals from Non-Uniform Background Fluorescence Savannah J. West1, Chase Hoffman2, Naga S. Annamdevula2, Kenny T. Trinh3, Thomas C. Rich2, Silas J. Leavesley3. 1 Biomedical Sciences, University of South Alabama, Mobile, AL, USA, 2 Pharmacology, University of South Alabama, Mobile, AL, USA, 3Chemical and Biomolecular Engineering, University of South Alabama, Mobile, AL, USA. In recent years Fo¨rster resonance energy transfer (FRET) has become a standard imaging approach to gain insight into localized biochemical processes within cells. These processes include changes in protein colocalization, second messenger concentration, and activation of effector proteins such as protein kinase A (PKA). However, there are several limitations in FRET measurements that are not often considered. In recent years our group has used hyperspectral imaging approaches to address a subset of these limitations, including the inherently low signal-to-noise ratio of standard two and three filter set FRET measurements. Here we present data demonstrating that hyperspectral imaging approaches can be used to correct for non-uniform background fluorescence in low intensity FRET measurements. We utilized the FRET-based cAMP probe H188. The H188 probe contains a cAMP binding domain between donor (Turquoise) and acceptor (Venus) fluorescent proteins. The probe was transfected into pulmonary microvascular endothelial cells plated on glass coverslips. Hyperspectral image stacks were acquired using a Nikon A1R inverted confocal microscope. Changes in cAMP were triggered by addition of 0.1 mM isoproterenol (a beta adrenergic receptor agonist) 0.1 mM PGE1 (a prostanoid receptor agonist) or 50 mM forskolin (an adenylyl cyclase activator). Data were analyzed using Nikon Elements software and custom MATLAB scripts. Results demonstrate that non-uniform background fluorescence associated with glass coverslips can contaminate FRET measurements from weak fluorescence signals. Linear unmixing approaches were able to separate the abundances of background, donor and acceptor fluorescence signals. Thus, results from this study demonstrate that hyperspectral unmixing approaches can readily separate nonuniform background fluorescence from other fluorescence signatures, allowing for more accurate quantification of FRET efficiency. This work was supported by NIH P01HL066299, NIH S10RR027535, NIH T32HL076125, AHA

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16PRE27130004, USA SURF Program, and the Abraham Mitchell Cancer Research Fund. 2228-Pos Board B548 Fluorescent Proteins for Super-Resolution Microscopy Karin Nienhaus1, Gerd U. Nienhaus1,2. 1 Institute of Applied Physics, Karlsruhe Institute of Technology, Karlsruhe, Germany, 2University of Illinois at Urbana-Champaign, Urbana, IL, USA. Super-resolution fluorescence microscopy is the method of choice to monitor cellular and subcellular biological processes in live cells. Among the different fluorescent labels presently available, fluorescent proteins (FPs) of the GFP family have the key advantage of being genetically encodable. Localizationbased super-resolution microscopy approaches require photoactivatable FPs (PA-FPs) that will change their spectral properties upon irradiation with light of a particular wavelength. To be able to distinguish individual, activated fluorophores from the background and to localize them with high precision, a high photon yield in the activated state and a high dynamic range, i.e., the contrast ratio between the fluorescence of the activated (on) and deactivated (off) states, are essential. In stimulated emission depletion (STED) super-resolution microscopy, the sample is raster-scanned by a tightly focused excitation beam followed by a red-shifted, donut-shaped depletion beam. Any FP used for STED must be exquisitely photostable because it has to go through multiple excitation-depletion cycles while the sample is scanned near its location. Moreover, it must be excitable and de-excitable by the lasers that are typically installed in commercial STED microscopes. Therefore, far-red emitting FPs are preferred. We have selected the green-to-red photoconvertible mEosFPthermo and the far-red emitting mGarnet as templates for targeted protein engineering. Considering that FPs are all very similar and share the same scaffold, an obvious strategy was to identify specific amino acid residues that elicit certain properties to one FP and introduce the corresponding amino acid in the other FP variant by using site-directed mutagenesis. As we will show, such simple rational engineering approaches often do not meet with success, which clearly shows that our current understanding of the physics of proteins is far from being complete. 2229-Pos Board B549 Wide Scale Investigation of Protein Interactions by Automation of Fluorescent Polarization and Fluctuation Analysis Tuan A. Nguyen, Grace H. Taumoefolau, Youngchan Kim, Henry L. Puhl, Steven S. Vogel. NIAAA, NIH, Rockville, MD, USA. Monitoring changes in molecular conformation is essential for studying protein interactions within and between complexes. Methods such as FRET or FCS reveal limited aspects of these changes, which in many cases is insufficient for interpretation. Fluorescent Polarization and Fluctuation Analysis (FPFA), a time-correlated single-photon counting technique that combines homoFRET and FCS was developed to address this problem. In FPFA changes in protein complex mass and/or shape, the number of fluorescent subunits per complex, as well as subunit proximity (1 – 10 nm), is simultaneously detected. This multimodal approach was validated with series of 6 fluorescent oligomers composed of between 1 and 6 concatenated Venus molecules, and successfully employed to investigate structural dynamics of calcium/calmodulin-dependent protein kinase II (CaMKII) – a multimeric protein kinase that is enriched in synaptic spines and dendrites that is involved in memory and synaptic modulation, as well as microtubule-associated protein 1A/1B-light chain 3 (LC3) – a soluble protein that is a key component of autophagy, a homeostasis process responsible for the turnover of cellular components. Here we have extended FPFA method by developing a robotic microscope that can measure up to 96 samples automatically. This FPFA automation enables wide-scale biophysical analysis of protein-protein interactions, and hence facilitates the identification of drugs that target protein complexes as potential therapeutic sites for diseases of aggregation and abnormal protein-protein interactions. 2230-Pos Board B550 Comparison Study on Fluorescence Quenching Ability of DNA Wrapped Single- and Multi-Walled Carbon Nanotubes Shusuke Oura, Katsuki Izumi, Kazuo Umemura. Tokyo University of Science, Shinjuku-ku, Japan. In terms of superior physical properties of carbon nanotubes (CNTs), CNT has been intensively studied for various applications including biosensors and drug delivery. For medical applications, hybridization of single-stranded DNA (ssDNA) and CNT (ssDNA-CNTs) is key to utilize them multiply because DNA can bind to various biomolecules. For example, Li et al. used ssDNACNTs for nucleic acid detection by applying the reaction of fluorescent molecules and CNTs. It is known that, due to electron transfer, fluorescence