High Resolution Laser Microscopy: a fascinating method to explore the molecular world

Transcription

1 High Resolution Laser Microscopy: a fascinating method to explore the molecular world Alfred J. Meixner Physical and Theoretical Chemistry Laboratory University of Siegen

2 Single-molecule spectroscopy and nano-optics

3 Outline Confocal microscopy optical single-microscopy Fluorescence spectroscopy of single molecules SERRS spectroscopy of single molecules Single-molecule spectroscopy at 1.8 K Highly confined optical fields Lab. tour

4 Confocal Fluorescence Microscopy fluorescence excitation energy level diagram from computer x z y sample molecule S 1 from laser dichroic mirror T 1 notch filter I exc I fluo source pinhole image pinhole S 0 photon counter to computer

5 diffraction limited imaging: Single Molecule Confocal Microscopy and Resolution Limit spatial resolution:?? 2 x i? 1.22 NA confocal imaging:? x i?? x o M

6 Single Molecule Fluorescence Excitation Microscopy laser x z y sample molecule image pinhole spectrometer polarizing beam splitter x-pol.detector y-pol.detector

7 Polarization images of individual R630 - molecules Counts/5ms Counts/5ms 0 x-polarization 6 µm y-polarization 0

8 Dynamics: molecules in motion

9 Dynamics: orientation jumps?? R f? DE? D? E 2 y-polarization x-polarization

10 Dynamics: intensity fluctuations

11 Spectroscopy: distinguishing conformers 9-amino-N-(2,6-diisopropylphenyl) perylen-3,4-dicarboximid N,N-di(tert-butoxycarbonyl)-API Sample preparation: spin coated film of 20g/l polystyrene (MW=250,000) in toluene dye content M Blum, Stracke, Becker, Müllen, Meixner, J. Phys. Chem. A 105 (2001)

12 Single molecule spectra of API and DAPI DAPI O N O Characteristic sequences of single molecule fluorescence spectra; ensemble spectra in the back planes of the graphs 10 O O tbut API N O O tbut ? / nm 20 off-resonance conformer in-resonance conformer O N O NH ? / nm ? / nm 20

13 Conformation jumps of single API molecules in- to offresonance transition ? / nm ? / nm off- to inresonance transition ? / nm ? / nm Blum, Stracke, Becker, Müllen, Meixner, Chem. Phys. Lett. 325 (2000)

14 Spectroscopy: amino-chromophore-resonance H H N H H N API + H(TFA) H(API) + + TFA - O. D Fluorescence / (a. u.) [TFA] = 51,9 mm [TFA] = 6,5 mm [TFA] = 13,0 mm [TFA] = 26,0 mm [TFA] = 0,0 mm ? / nm ? / nm Extinction spectra of API in Acetonitrile with increasing TFA concentration Fluorescence spectra of API in Acetonitrile with increasing TFA concentration

15 Surface enhanced resonance Raman scattering vs. fluorescence emission? Raman ~ cm 2 =>? SERRS ~ cm 2 Er(t)??tot wavelength [nm] Es(t)

16 Absorption and Fluorescence v Resonance Raman Scattering v S 1 I L (? L ) I F (? F ) S 1 I L (? L ) I fi (??) i f S 0 i f S 0

17 Rhodamine 6G SERRS spectrum 1s/spectrum 618 C-C-C ring i.p. bend 776 C-H o.p. bend 1137 C-H i.p. bend 1270 C-O-C str arom. C-C str arom. C-C str arom. C-C str arom. C-C str combinations

18 Sample preparation Lee and Meisel, JPC 86 (1982) 3391 Colloidal solution: Ag-particles ~ M dye: Rhodamine 6G ~ 10-9 M ~ M ~ M incubation: ~ 12 h 500 nm

19 Towards the single-molecule limit 10-9 M dye concentration scattered excitation light? = nm scattered light at? > nm ) 2) 3) Meixner, Vosgröne, Sackrow, J. Lumin (2001)

20 Single-molecule SERRS spectra - inhomogeneous spectral behavior - intensity fluctuations, blinking - splitting of spectral lines - spectral diffusion - inhomogeneous line broadening - towards the homogeneous linewidth

21 Towards homogeneous line broadening spectrometer resolution???? 4 cm -1 narrowest linewidth???? 7.5 cm -1 estimate for minimum coherence time?t = 750 fs

22 Single-molecule rhodamine 6G SERRS spectra 1s/spectrum, M,? exc = nm time/sec wavenumbers / cm-1

23 Confocal Microscope for Cryogenic Temperature Features: immersed in liq. He (1.8K) confocal/near-field parabolic mirror objective confocal resolution: diffraction limited nfo-resolution: physics will show shear-force topography

24 Heart of the parabolic mirror microscope

25 scanner Parabolic mirror

26 Single Terrylene molecules in Octadecane at 1.8K confocal fluorescence excitation spectra four molecules wide field confocal two molecules Drechsler, Lieb, Debus, Meixner Opt. Express 9 (2001)

27 Fluorescence spectrum of a single Terrylene molecule at 1.8K Drechsler, Lieb, Debus, Meixner,Opt. Express 9 (2001)

28 Focusing with linear polarized light z f 0 y x k Lieb, Meixner, Opt. Express 8 (2001)

29 Focusing with radially polarized light z f 0 y y x x kk E Lieb, Meixner, Opt. Express 8 (2001)

30 Field enhancement by an optical half-wave antenna (field enhancement calculated by L. Novotny, Rochester) f 0 y x x calculation:? = 800 nm tip radius = 10 nm dielectric sample enhancement 10 4 kk E bar = 10?m bar = 100 nm

31 Femtosecond microscopy lab.

32 Femtosecond laser with microscope

33 Confocal / near-field microscope

34 Acknowledgment co-workers: P. Anger Ch. Blum J. Bonse Ch. Debus A. Drechsler R. Gallacchi H. Kneppe A. Lieb R. Neidhardt M. Sackrow F. Schleifenbaum G. Schulte F. Stracke former co-workers: M. A. Bopp (Basel) G. Tarrach (Basel) M. Weber (Siegen, Dresden) Dye molecules: K. Müllen, S. Becker (Mainz) K. H. Drexhage (Siegen) Zeolites G. Calzaferri (Bern) S. Megelski (Bern) Ag nano-particles: W. Plieth(Dresden) G. Sandman (Dresden) P. Moyer (Charlotte) Funding University of Siegen NRW DFG VW-Stiftung Swiss Nat. Sci. Fund. ETH