High-Resolution Imaging of Single Fluorescent Molecules with the Optical Near-Field of a Metal Tip H. G. Frey, S. Witt, K. Felderer, and R. Guckenberger, Phys. Rev. Lett. 93, 200801 (2004). Marc McGuigan Journal Club Monday, April 10, 2006 Outline Introduction Near Field Microscopy Purpose Experimental Setup Sample Preparation Results Data Model Conclusion Beating the Diffraction Limit d min 2 NA d min visible 0.2 0.5m Alternatives •Scanning Tunneling Microscope •Atomic Force Microscope •Scanning Electron Microscope •Transmission Electron Microscope Why use visible light? •Contrast •Easier Sample Preparation History (1) 1928 – Synge Idea Strong light source behind thin metal film 100 nm diameter hole to illuminate biological sample Sample less than 100 nm away from source (2) Discusses ideas in letters to Albert Einstein (3) 1972 – E. A. Ash and G. Nicholls Passed microwaves (3 cm) through 1.5 mm aperture Scanned over grating and were able to resolve 0.5 mm lines and 0.5 mm gaps in grating 1984 Pohl, Denk, Duerig (IBM) (SNOM) Lewis group (Cornell) (NSOM) Subwavelength aperture at apex of sharp transparent probe tip that is coated with metal Diagram Source: Molecular Expressions Optical Microscopy Primer, http://micro.magnet.fsu.edu/primer/index.html Evanescent Waves Wave vectors propagating in k space Total Internal Reflection n1 sin 1 n2 sin 2 n2 n 1 c sin 1 k k n1k 2 1x 2 1z 2 k22x k22z n2k 2 (4) E1 ei k1x xk1z z t E2 ei k2 x xk2 z z t n cos 2 1 n2 E2 e in1k 2 n2 sin 2 1 n1 n2 n1 2 sin 2 1 x sin 1 z it k1z k 2 z n1k sin 1 E2 exi z t k2 x n2k cos2 n n1k sin 1 2 n1 Diagram Source: K. Iizuka, Elements of Photonics: In Free Space and Special Media, Volume 1 (John Wiley & Sons, New York, 2002). 2 2 Evanescent Waves Evanescent Waves on a Corrugated Metal Surface kx2 kz2 nk Evanescent Waves on an Array of Metal Pins 2 To satisfy boundary conditions: zm 2 2 2 k xn m d 2 md k zm This can be re-written as: 2 The value of kxn is imaginary for high values of m and the waves are evanescent waves d m dc 2 Above dc kx is always imaginary and all the waves in x are evanescent waves. Diagram Source: K. Iizuka, Elements of Photonics: In Free Space and Special Media, Volume 1 (John Wiley & Sons, New York, 2002). Modes of Near Field Imaging Different types of scanning near field optical microscopes NSOM Configurations (c) collection/illumination (a) collection (a) Aperture NSOM (b) illumination (d) oblique collection (b) Apertureless NSOM (e) oblique illumination (f) Dark field Diagram (left) Source: M. A. Paesler and P. J. Moyer, Near Field Optics: Theory, Instrumentation, and Applications (John Wiley & Sons, New York, 1996). Diagram (right) Source: B. Hecht et al., J. Chem. Phys. 112, 7761 (2000). (c) Scanning tunneling optical microscope NSOM Setup Standard NSOM Setup (a) Illumination Tips (5) •Heating and pulling method - Optical fiber is heated with CO2 laser and pulled on both sides of heated area •Chemical etching method - Hydrofluoric acid used to etch glass fiber •Fiber coated with metal •Nanoparticle (Tip Enhanced) (b) Collection and Redistribution (c) Detection Diagram (left) Source: B. Hecht et al., J. Chem. Phys. 112, 7761 (2000). Diagram (right) Source: Molecular Expressions Optical Microscopy Primer, http://micro.magnet.fsu.edu/primer/index.html Aperture NSOM Resolution: 50-100 nm Aluminum-coated aperture probes Problems (6) 300 nm (a), (b) prepared by pulling (c), (d) prepared by etching 300 nm (a), (c) macroscopic shape, SEM and optical image (b), (d) SEM close-up of the aperture region • Difficult to create smooth aluminum coating on nanometer scale • Flat ends of the probes are not good for high resolution topographic imaging • Absorption of light by metal coating causes significant heating Diagram (left) Source: B. Hecht et al., J. Chem. Phys. 112, 7761 (2000). Diagram (right) Source: Molecular Expressions Optical Microscopy Primer, http://micro.magnet.fsu.edu/primer/index.html Tip-Enhanced NSOM Schematic of experimental setup for tip-enhanced near field Induced surface charge density in metal probe Left: Incident wave polarized perpendicular to tip axis Right: Incident wave polarized along tip axis Resolution: 10-20 nm The incident field should be polarized along the tip axis to maximize field enhancement Need large near field enhancement so the signal can be detected in the far field Causes for Enhanced Electric Field: (7) •Electrostatic lightning rod effect (depends on geometry) •Surface plasmon resonances (depend on excitation wavelength and geometry) Diagram (left) Source: A. Hartschuh, M. R. Beversluis, A. Bouhelier, and L. Novotny, Phil. Trans. R. Soc. Lond. A. 362, 807 (2004). (7) Diagram (right) Source: L. Novotny, R. X. Bian, and X. S. Xie, Phys. Rev. Lett. 79, 645 (1997). (8) NSOM and Fluorescence Aperture NSOM resolution ~ 50 nm Simultaneous topographic image (a) and near-field twophoton excited fluorescence image (b) of J-aggregates of PIC dye in PVS film on a glass substrate. Tip-enhanced •better resolution •high background signal •Bleaching of dyes One solution: two-photon excitation Two-photon excitation is a nonlinear process Detected signal is proportional to the square of the intensity enhancement factor (6) Illuminated area of sample: S = 105 nm2 Intensity enhanced area under tip: σ = 100 nm2 2 Signal f f 1000 1000 Noise S Diagram (left) Source: E. J. Sanchez, L. Novotny, and X. S. Xie, Phys. Rev. Lett. 82, 4014 (1999). Purpose Interest: “investigation of self-fluorescing or fluorescence labeled macromolecules at the single molecule level.” Challenge: combine optical and topographical resolution of NSOM with fluorophore sensitivity Results: “highly resolved optical imaging of single dyes” “high-resolution topographs” “tip-on-aperture” probe Drawing of “tip-on-aperture probe with the DNA sample. Thin optical fiber in etching solution (10) Tip covered with Cr (for adhesion) then 200 nm Au for contrast in SEM Focus electron beam of SEM on the center of the aperture Electron-beam-deposited tip (EBD) formed (7 s, 8 kV) SEM images of a “tip-on-aperture” probe 3.5 nm Cr and 33 nm Al deposited by evaporation at 45o (a) Before metallization Diagram (left) Source: H. G. Frey, F. Keilmann, A. Kriele, and R. Guckenberger, Appl. Phys. Lett. 81, 5030 (2002). (b) After metallization Experimental Setup Light Source: argon laser (514 nm) Light coupled to glass fiber onto the sample Light transmitted through the sample and collected by objective (0.95 NA) on inverse light microscope Light filtered by 550 nm long pass filter Signal detected by APD Sample scanned ~ 1 micron per second Scanned at constant distance with shear force feedback Polarization of incident laser light adjusted to optimize S/N 1/3 of probes provide good fluorescence results Sample Preparation DNA Cy-3 fluorophores covalently bound to the termini of DNA Samples prepared in a polymerase chain reaction (PCR) Mica Sheets 20 μl of 400 mM NiCl solution in water 2 min later – solution bottled off and 30 μl drop of DNA (with Cy-3 label) solution applied to the sheet 10 min later – washed in ultrapure water and dried with nitrogen 2 Results Fluorescence image of single Cy-3 dye molecules, which appear mostly as double maxima. 200 nm Fitted tip radius: 12 nm FWHM = 10 nm 25 nm Zoomed image of a dye molecule together with a section along the 25 nm line (three lines average). 25 nm Enlarged image of a 25 nm bleaching event from one scan line (oriented vertically) to the next one. Data Model •Dye molecule excitation proportional to squared field component parallel to dipole moment •They believe that the field from the aperture light does not substantially influence the experiment Etip 10Eaperture •Dye dipoles oriented vertically experience maximum excitation directly below the tip •Dye oriented in sample plane displays two symmetric maxima •Inclined dyes display asymmetric peaks •Vertical dye under the tip displays a circular structure Data Model Quantum yield d Software: MATHLAB 6.5 (Mathworks) Classical Mirror Image Calculation Neglected: Retroaction of dye dipole on tip dipole Retardation effects Emission in direction of objective used to calculate final signal Al 44.7 15.0i mica 2.56 Lifetime without mirror 3qc3 1 ImE0 3 20 n1 Index of refraction of medium with dipole •Fit Parameters •X, Y position of dye •3D orientation of dye •Normalization factor for dye brightness and local background •Parameters assumed constant •Tip radius •Tip-sample distance •Quantum efficiency = 0.3 Results with Data Model Fluorescence patterns of differently tilted dye molecules. Measurements Image size: 117 nm Fitted tip radius: 22nm Patterns calculated with parameters fitted to the measurements Tilt angle: 0o Tilt angle: 14o Tilt angle: 20o Tilt angle: 49o Tilt angle: 68o •Tip-dye distance (calculated): 1 nm •Tip-dye distance (approach curve): 2-3 nm •Why the discrepancy? •Treatment of quenching effects neglects contributions with a stronger dependence on distance becoming important within 5 nm •Tip apex flatter than a sphere •Moon-like and ring-like patterns due to strong quenching effects when tip-dye distance below 3 nm •As tip-dye distance increases central minimum decreases in size •Total number of photon counts per pattern decreased by factor of 2 when tipdye distance increases by 5 nm Results DNA with Cy-3 labeled termini on mica and corresponding data modeling. (a) Topography together with calculated positions of analyzed dye molecules (b) Fluorescence image Note: A fitted parabola has been subtracted from each scan line to flatten the data Accuracy in dye positions: 0.5 nm standard deviation Analyzed dye molecules (c) Positions of dye molecules in (a) with tilt angles (upper number) and azimuth angles (lower numbers) from first approximation fits in (d) Fitted tip radius: 12 nm (d) First approximation fits Green: Good fits Yellow: Problematic fits 200 nm Azimuth angle accurate to 5o Accuracy of tilt angle better than 10o Results Fluorescence pattern of two dyes located close to each other Single dye molecule 300 nm 300 nm (a) Experimental data (b) Best fit when assuming two dyes for the encircled pattern (c) Difference between data (a) and fit (b) Conclusion Results Near-field optical image of single fluorescent dye molecules at high resolution High resolution topographic image of dye molecules Improvements Optimize tip-aperture geometry to allow plasmon resonance Vary tip length Change material Sharpen the metal tip to improve resolution References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. B. Hecht et al., J. Chem. Phys. 112, 7761 (2000). M. A. Paesler and P. J. Moyer, Near Field Optics: Theory, Instrumentation, and Applications (John Wiley & Sons, New York, 1996). Molecular Expressions Optical Microscopy Primer, http://micro.magnet.fsu.edu/primer/index.html K. Iizuka, Elements of Photonics: In Free Space and Special Media, Volume 1 (John Wiley & Sons, New York, 2002). P. N. Prasad, Nanophotonics (John Wiley & Sons, Hoboken, 2004). E. J. Sanchez, L. Novotny, and X. S. Xie, Phys. Rev. Lett. 82, 4014 (1999). A. Hartschuh, M. R. Beversluis, A. Bouhelier, and L. Novotny, Phil. Trans. R. Soc. Lond. A. 362, 807 (2004). L. Novotny, R. X. Bian, and X. S. Xie, Phys. Rev. Lett. 79, 645 (1997). N. Anderson, A. Bouhelier, L. Novotny, J. Opt. A. 8, S227 (2006). H. G. Frey, F. Keilmann, A. Kriele, and R. Guckenberger, Appl. Phys. Lett. 81, 5030 (2002).