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High High- Pressure Biophysics and Biotechnology Pressure Biophysics and Biotechnology at the Department of Physics: 2008 Update at the Department of Physics: 2008 Update Paul Urayama (39 Culler, Paul Urayama (39 Culler, [email protected] [email protected]), Miami University, Oxford, OH 45056 ), Miami University, Oxford, OH 45056 Funding Acknowledgements Research Corporation; College of Arts and Science, Miami University; Undergraduate Summer Scholars Program, Miami University. Fall 2008 Poster Seminar, Miami University, Dept. of Physics. September 3, 2008. Lab Highlights (Fall 2007 Lab Highlights (Fall 2007 – Summer 2008) Summer 2008) Papers (peer reviewed) Papers (peer reviewed) Undergraduate authors underlined ; graduate authors italicized. • E.W. Frey , Sara R. Savage , P. Urayama. “High-pressure, fluorescence-based sensing of calcium ions.” (in preparation, 2008). • H.M. DePedro, P. Urayama. “Using LysoSensor Yellow/Blue DND-160 to sense acidic pH under high hydrostatic pressures.” (Analytical Biochemistry, submitted 2008). • S.B. Keller , J.A. Dudley , K. Binzel , J. Jasensky , H.M. DePedro, E.W. Frey , P. Urayama. “A calibration approach for rapid fluorescence lifetime determination for applications using time-gated detection and finite pulse width excitation,” Analytical Chemistry, in press (2008). • P. Urayama, E.W. Frey , M.J. Eldridge. A fluid handling system with finger-tightened connectors for biological studies at kilo- atmosphere pressures, Review of Scientific Instruments, 79: 046103 (2008). • T. Haver, E.C. Raber, P. Urayama. An application of spatial deconvolution to a capillary-based high-pressure chamber for fluorescence microscopy imaging, Journal of Microscopy, 230: 363-371 (2008). Abstracts (refereed) Abstracts (refereed) Undergraduate authors underlined ; graduate authors italicized. • P. Urayama, E.W. Frey , S.R. Savage . “High-pressure, fluorescence-based sensing of calcium ions.” Fifth International High Pressure Bioscience and Biotechnology Conference, LaJolla, CA, September 15-20, 2008. • E.W. Frey and P. Urayama. Fluorescence-based calcium-ion sensing at high hydrostatic pressures. 2008 Biophysical Society Meeting Abstracts. Biophysical Journal, Supplement: 326a (2008). Awards and Recognitions (selected highlights) Awards and Recognitions (selected highlights) 2008-08-18: Work by Thomas Haver and Erica Raber is featured in the "Technology Solutions" section of the August 2008 Biophotonics International. 2008-07-01: A research proposal to study high-pressure cellular metabolism was funded by Research Corporation. 2008-05-05: Eric Frey and Michael Eldridge's paper has been chosen for inclusion in the April 15, 2008, edition of the Virtual Journal of Biological Physics Research. 2008-04-30: Eric Frey was awarded the Outstanding Senior and the Outstanding Undergraduate Researcher awards. Hector Michael DePedro was awarded the Outstanding Graduate Researcher award. Josh Jasensky, Scott Keller, and Michael Maffett all received department Commendation in Undergraduate Research awards. 2008-02-13: Josh Jasensky have been selected to be a 2008 Undergraduate Summer Scholar. 2007-12-13: Eric Frey has been awarded a Sigma Xi Grants-in-Aid of Research, a nationally competitive program with a 20% funding rate! 2007-11-08: Eric Frey was awarded a Student Travel Award to the 2008 Biophysical Society Annual Meeting. High High-pressure, fluorescence pressure, fluorescence-based sensing of calcium ions based sensing of calcium ions Sara Savage ( Sara Savage (’09), Eric W. Frey ( 09), Eric W. Frey (’08) 08) To be presented at the 5th International Conference on High Pressure Bioscience and Biotechnology, La Jolla, CA Sept 15-20, 2008. Conference Abstract Because calcium ions are nearly ubiquitous in cellular signaling and control, methods for calcium-ion sensing at high pressures are valuable in investigating pressure effects on cellular function. Optically-active, calcium-sensitive dyes provide the means for quantitative ion sensing at physiologically-relevant ion concentrations. Here we present high- pressure techniques for calcium-ion sensing using the Fluo-class of intracellular dyes. The dyes (< 2μM) were characterized in EGTA/MOPS calcium-buffer solutions (10mM EGTA, 30mM MOPS, 100mM KCl, pH7.2, pCa 4 through 9) with fluorescence emission spectra measured at pressures up to 500 atm on a custom-built spectrofluorimeter system utilizing a quartz-capillary pressure chamber. The dye’s pK under pressure was determined using a two-state bound/unbound model for fluorophore/calcium ion dissociation. Assuming an Arrhenius relation, the effective ΔV of the calcium dissociation reaction was determined. We find that corrections related to the dye’s ΔV (i.e., pressure-dependent pK) are important to consider when making quantitative measurements because the ΔV values, which are in the 10- ml/mol range, are comparable to that of a wide range of biochemical reactions. In addition to calcium sensing in biological systems, quantitative results presented here have potential applications in the broader high-pressure ion- sensing field. pCa 4 5 6 7 8 9 10 intensity (arb) pressure (atm) 0 100 200 300 400 500 600 intensity (arb) pCa(1 atm) = 7.12 in MOPS/EGTA calcium buffer FIGURE 1: Ambient- pressure fluorescence emission intensity of the calcium-sensitive dye Fluo4 versus free calcium concentration. The red line is a fit to a two-state (calcium bound/unbound) model. λ(ex) = 500nm; 2μM Fluo4 in a MOPS/EGTA calcium buffer. FIGURE 2: Emission intensity decreases upon pressurization due to a change in the calcium- binding properties of the dye. Such information is used to find the thermodynamic ΔV of the calcium-binding reaction. See Salerno et al. (Analytical Biochemistry, 362: 258-267, 2007) for more information. Josh and Junaid aligning the laser system. Sara preparing the high- pressure setup for an experiment. Lab Members (l to r) Junaid Farooqi (graduate student) Joshua Jasenski (physics) Brian Casto (engineering physics) Sara Savage (zoology) Not shown, Lauren Regueyra, Jordan Ryan. Congratulations to 2008 Graduates! (l to r) Hector DePedro (MS, Physics) Scott Keller (BS, Eng. Physics) Daniel Horne (BS, Eng. Physics) Eric Frey (BS, Physics) Not shown, Michael Maffett (BS, Eng. Physics). A calibration approach for rapid fluorescence lifetime A calibration approach for rapid fluorescence lifetime determination for applications using time determination for applications using time-gated detection gated detection and finite pulse width excitation and finite pulse width excitation Scott B. Keller ( Scott B. Keller (’08), Jonathan A. Dudley ( 08), Jonathan A. Dudley (’07), Katherine 07), Katherine Binzel Binzel (’07), 07), Joshua Joshua Jasensky Jasensky (’09), Hector Michael 09), Hector Michael DePedro DePedro (GS (GS ‘08), Eric W. Frey 08), Eric W. Frey (’08) 08) in press, Analytical Chemistry, 2008. Paper Abstract Time-gated techniques are useful for the rapid sampling of excited-state (fluorescence) emission decays in the time domain. Gated detectors coupled with bright, economical, nanosecond-pulsed light sources like flashlamps and nitrogen lasers are an attractive combination for bioanalytical and biomedical applications. Here we present a calibration approach for lifetime determination that is non-iterative and that does not assume a negligible instrument response function (i.e., a negligible excitation pulse width) as does most current rapid lifetime determination approaches. Analogous to a transducer-based sensor, signals from fluorophores of known lifetime (0.5 – 12 ns) serve as calibration references. A fast avalanche photodiode and a GHz-bandwidth digital oscilloscope is used to detect transient emission from reference samples excited using a nitrogen laser. We find that the normalized time-integrated emission signal is proportional to the lifetime and can be determined with good reproducibility (typically <100 ps) even for data with poor signal-to-noise ratios (~20). Results are in good agreement with simulations. Additionally, a new time-gating scheme for fluorescence lifetime imaging applications is proposed. In conclusion, a calibration-based approach is a valuable analysis tool for the rapid determination of lifetime in applications using time-gated detection and finite pulse width excitation. Figure 1. Top, IRF’s used for the simulations, including Gaussian (circle), Lorentzian (inverted triangle), rectangular (square), and triangular (upright triangle) IRF forms. A measured IRF from the nitrogen laser is also shown (solid line). Peaks have been centered at t = 0. Middle, simulation results showing area versus τ/IRFFWHM for the four IRF forms (same symbols as the top figure). Results are linear for a wide range of τ/IRFFWHM values and IRF forms. Areas have been normalized to the τ/IRFFWHM = 0 value. The percent deviation is a deviation from linearity calculated from a linear fit to the entire plot. Bottom, detail of the small- τ/IRFFWHM simulation region (same symbols as the top figure). Lines are linear fits to the entire plot. Note how both positive and negative deviations from an overall linearity are possible. Figure 2. Top, measured transient emission from the reference fluorophores using UV excitation. Signals have been background subtracted, scaled, and the time axis shifted so that t = 0 corresponds to 50% maximum intensity. Samples, from the smallest to largest FWHM, are the IRF, POPOP in ethanol, rhodamine B in water, rhodamine B in ethanol, and 9- cyanoanthracene in ethanol. The plot for the 9-cyanoanthracene sample has been truncated for clarity. The SNR is ~20. Bottom, comparison of reference areas with simulation. The best-fit line is shown, and is intermediate to simulation results for the Gaussian (circle) and Lorenztian (inverted triangle) IRF. Highly linear behavior demonstrates that the area is a useful parameter on which to base the calibration. Figure 1. Chamber for capillary imaging. Samples are housed in the capillary, visible through the central bore in the plates. The plates are 3" x 1", the size of a standard microscope slide. Figure 2. Images of 0.7 μm fluorescent microspheres (40x, 1.3NA objective) before and after deconvolution. Left, raw image taken inside of a capillary; middle, image deconvolved using a theoretical PSF designed for standard slide imaging; right, image deconvolved using the measured PSF from Figure 2. Deconvolution using a PSF that neglects capillary curvature (middle) gives results comparable to using a measured PSF (right). The scale bar is 5 μm. As featured in the "Technology Solutions" section of the August 2008 Biophotonics International. Methanol Methanol-denaturated dynamics of NADH probed using denaturated dynamics of NADH probed using fluorescence spectroscopy fluorescence spectroscopy Joshua Joshua Jasensky Jasensky (’09, USS), 09, USS), Junaid Junaid Farooqi Farooqi (GS (GS ’09), Daniel Horne 09), Daniel Horne (’08) 08) To be presented at OSAPS, Oct 10-11, 2008. NADH is an metabolic co-factor useful for non-invasively probing cellular metabolic activity. NADH folding- unfolding equilibrium and intermolecular NADH interactions in solution can be probed using fluorescence spectroscopy. Future studies involve solution and in vivo investigations under pressure. FIGURE 2. Top, NADH folded and unfolded states. Bottom, emission peak wavelength versus denaturant concentration. Fit is to a linear model for solvent denaturation. ΔG = ΔG + m [denaturant] m = 560 J/M Conclusion: thermodynamic parameters can be measured using fluorescence spectroscopy. FIGURE 1. Top, emission spectra of NADH under folded (0% MeOH) (left) and unfolded (90% MeOH) (right) conditions at 0.8 μM (blue) and 100 μM (red) NADH. 337nm ex, pH 7.5 MOPS buffer solution. Bottom, peak NADH emission wavelength under folded (red) and unfolded (blue) conditions. Conclusion: concentration dependence evidences greater sensitivity of the unfolded state to intermolecular ring interactions. For For Students Students If you are thinking about graduate or professional school, or would like hands-on experience doing exciting science, read on… An important goal of my laboratory is to ACTIVELY involve students in the research process. You are encouraged to initiate and develop projects, and are given the freedom and guidance to do so. The earlier you get involved and STAY involved, the more substantial and meaningful the experience will be. See me if interested. Prof. Paul Urayama 39 Culler Hall [email protected] 529.9274
