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(12) (10) Patent No.: US 7,956,989 B2 Gryczynski et al. (45) Date of Patent: Jun. 7, 2011 (54) SURFACE PT-ASMON ASSISTED 20067/l)“22764’g‘l‘9t 221: lggggg at al ~~~~~~~~~~~~~~ ~~ gggfelg omsen et a . MICROSCOPE 2009/0079978 Al * 3/2009 Kimura ....................... .. 356/301 (75) Inventors: Zygmunt Gryczynski, Fort Worth, TX OTHER PUBLICATIONS (US); Ignac_y Gryczynskl’ Fort Worth’ Borejdo, J ., et al., “Application of surface plasmon coupled emission TX (US); N115 Calandefi Gothenburg to study of muscle.” Biophys J (2006), 91(7):2626-35. (SE); Julian B0l‘ejd0, Dallas, TX (US) Borejdo, J., et al., “Fluorescence Correlation Spectroscopy in Surface Plasmon Coupled Emission Microscope.” Optics Express (2006), (73) Assignee: University of North Texas Health ]Ta4(1;)17§7§['71§33~ 1 “I S_ Fl P _ I _ _th at 7» W°“h> TX (US) Biophys J (2006) 9014662-4671. py’ Calander, N., Theory and simulation of surface plasmon-coupled ( * ) Notice: Subject to any disclaimer, the term ofthis directional emission from fluorophores at planar structures. Anal patent is extended or adjusted under 35 Chem (2004), 76(3)12163'73~ (Continued) (21) APPT NOJ 12/0181107 Primary Examiner — Michael P Stafira _ (74) Attorney, Agent, or Firm — Chalker Flores, LLP; (22) Filed: Jan. 22, 2008 Edwin S_ Flores (65) Prior Publication Data (57) ABSTRACT US 2008/0231834 A1 Sep. 25, 2008 The present invention includes a microscope and a method for using the microscope for single molecule with reduced phoRelated U,S, Application Data tobleaching of a fluorophore (20) that includes a light trans_ _ _ _ lucent material (16); a metal layer (18) disposed on the light (60) Provisional application No. 60/881,645, filed on Jan. translucent material (16); a medium (15) disposed on the 22, 2007 metal layer (18), the medium (15) having one or more fluorophores (20) capable of binding a target analyte (e.g., inside (51) Int CL a cell); a microscope positioned to observe the surface plasG01N 1/00 (2006.01) mon emissions from the one or more fluorophores (20) within (52) U.S. Cl. ....................................................... .. 356/36 50 _nan_0meters of the surface ef the metal layer (18); an (58) Field of Classification Search ......... .. 356/4454448 excltatlen some Capable of eX°1t1I1g_t1_1e one or more fivefoSee application file for Complete Search history phores (20), the excitation source positioned to strike the light translucent material (16) at a first angle; and a light detector (56) References Cited (38) that selectively detects emitted light generated by excited
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fluorophores (20) at a second angle (22), wherein light emitted by the one or more fluorophores (20) at the surface plasmon angle is detected through the microscope, such that single molecules may be detected without significantly degrading fluorophore (20) emissions.
36 Claims, 10 Drawing Sheets
OTHER PUBLICATIONS Gryczynski, I., et al., “Radiative decay engineering 4. Experimental
studies of surface plasmon-coupled directional emission.” Anal Biochem (2004), 324(2): 170-82.
Gryczynski, I., et al., “Surface plasmon-coupled emission using gold film.” J. Phys. Chem. B (2004), 108112568-12574.
Gryczynski, Z ., et al., “Minimization of detection volume by surfaceplasmon-coupled emission.” Anal Biochem (2006), 356(1): 125-31. Lakowicz, J . R., Radiative decay engineering: biophysical and biomedical applications. Anal Biochem (2001), 298(1):1-24. Lakowicz, J. R., et al., “Intrinsic fluorescence from DNA can be enhanced by metallic particles.” Biochem Biophys Res Commun (2001), 286(5):875-9.
Lakowicz, J . R., et al., “Radiative decay engineering. 2. Effects of Silver Island films on fluorescence intensity, lifetimes, and resonance energy transfer.” Anal Biochem (2002), 301(2):261-77.
Lakowicz, J . R., et al., “Directional surface plasmon-coupled emission: A new method for high sensitivity detection.” Biochem Biophys Res Commun (2003), 307(3):435-9.
Lakowicz, J. R., Radiative decay engineering 3. Surface p1asmoncoupled directional emission. Anal Biochem (2004), 324(2): 153-69.
Malicka, J ., et al., Increased resonance energy transfer between fluorophores bound to DNA in proximity to metallic silver particles.” Anal Biochem (2003), 315(2): 160-9.
Malicka, J., et al., “Use of surface plasmon-coupled emission to measure DNA hybridization.” J Biomol Screen (2004), 9(3):208-15. Malicka, J ., et al., “Effects of metallic silver island films on resonance energy transfer between N,N’ -(dipropyl)-tetramethy1indocarbocyanine (Cy3)- and N,N’-(dipropyI)-tetramethylindodicarbocyanine (Cy5)-labeled DNA.” Biopolymers (2003), 70(4):595-603.
Matveeva, E., et al., “Metal-enhanced fluorescence immunoassays using total internal reflection and silver island-coated surfaces.” Anal Biochem (2004), 334(2):303-11.
miRBase: : Sequences. http://microma.sanger.ac.u1dcgi-bin/sequences/browse.pl (Jul. 31, 2007).
Muthu, P., et al., “Decreasing photobleaching by silver island films: application to muscle.” Anal Biochem (2007), 366(2):228-36. Neogi, A., et al., “Coupling of spontaneous emission from GaN-AIN quantum dots into silver surface plasmons.” Opt Lett (2005), 30(1):93-5.
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