High Resolution Laser Microscopy: a fascinating method to explore the molecular world Alfred J. Meixner Physical and Theoretical Chemistry Laboratory University of Siegen
Single-molecule spectroscopy and nano-optics
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
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
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
Single Molecule Fluorescence Excitation Microscopy laser x z y sample molecule image pinhole spectrometer polarizing beam splitter x-pol.detector y-pol.detector
Polarization images of individual R630 - molecules 400 400 Counts/5ms Counts/5ms 0 x-polarization 6 µm y-polarization 0
Dynamics: molecules in motion
Dynamics: orientation jumps?? R f? DE? D? E 2 y-polarization x-polarization
Dynamics: intensity fluctuations
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 10-11 M Blum, Stracke, Becker, Müllen, Meixner, J. Phys. Chem. A 105 (2001) 6983-6990
Single molecule spectra of API and DAPI DAPI O N O 750 500 250 0 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 550 650 750? / nm 20 off-resonance conformer in-resonance conformer 225 90 O N O 150 75 60 30 0 0 10 10 NH 2 550 650 750? / nm 20 550 650 750? / nm 20
Conformation jumps of single API molecules in- to offresonance transition 180 120 60 0 10 75 50 25 0 10 600? / nm 700 800 20 600? / nm 700 800 20 off- to inresonance transition 900 600 300 0 10 900 600 300 0 10 600? / nm 700 800 20 600? / nm Blum, Stracke, Becker, Müllen, Meixner, Chem. Phys. Lett. 325 (2000) 196-202 700 800 20
Spectroscopy: amino-chromophore-resonance H H N H H N API + H(TFA) H(API) + + TFA - O. D. 0.5 0.4 0.3 0.2 0.1 Fluorescence / (a. u.) [TFA] = 51,9 mm [TFA] = 6,5 mm [TFA] = 13,0 mm [TFA] = 26,0 mm [TFA] = 0,0 mm 0 400 450 500 550 600 650 700 750? / nm 500 550 600 650 700 750 800 850? / nm Extinction spectra of API in Acetonitrile with increasing TFA concentration Fluorescence spectra of API in Acetonitrile with increasing TFA concentration
Surface enhanced resonance Raman scattering vs. fluorescence emission? Raman ~ 10-29 cm 2 =>? SERRS ~ 10-14 cm 2 Er(t)??tot 400 500 600 700 wavelength [nm] Es(t)
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
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. 1366 arom. C-C str. 1509 arom. C-C str. 1575 arom. C-C str. 1650 arom. C-C str combinations
Sample preparation Lee and Meisel, JPC 86 (1982) 3391 Colloidal solution: Ag-particles ~ 10-11 M dye: Rhodamine 6G ~ 10-9 M ~ 10-11 M ~ 10-13 M incubation: ~ 12 h 500 nm
Towards the single-molecule limit 10-9 M dye concentration scattered excitation light? = 514.5 nm scattered light at? > 516.5 nm 1 2 3 1) 2) 3) Meixner, Vosgröne, Sackrow, J. Lumin 94-95 (2001) 147-152
Single-molecule SERRS spectra - inhomogeneous spectral behavior - intensity fluctuations, blinking - splitting of spectral lines - spectral diffusion - inhomogeneous line broadening - towards the homogeneous linewidth
Towards homogeneous line broadening spectrometer resolution???? 4 cm -1 narrowest linewidth???? 7.5 cm -1 estimate for minimum coherence time?t = 750 fs
Single-molecule rhodamine 6G SERRS spectra 1s/spectrum, 10-11 M,? exc = 514.5 nm time/sec wavenumbers / cm-1
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
Heart of the parabolic mirror microscope
scanner Parabolic mirror
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) 637-644
Fluorescence spectrum of a single Terrylene molecule at 1.8K Drechsler, Lieb, Debus, Meixner,Opt. Express 9 (2001) 637-644
Focusing with linear polarized light z f 0 y x k Lieb, Meixner, Opt. Express 8 (2001) 458-474
Focusing with radially polarized light z f 0 y y x x kk E Lieb, Meixner, Opt. Express 8 (2001) 458-474
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
Femtosecond microscopy lab.
Femtosecond laser with microscope
Confocal / near-field microscope
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