Single Molecule Spectroscopy and Imaging

Transcription

1 Single Molecule Spectroscopy and Imaging Ingo Gregor, Thomas Dertinger, Iris von der Hocht, Jan Sykora, Luru Dai, Jörg Enderlein Institute for Biological Information Processing 1 Forschungszentrum Jülich

2 Motivation Distribution functions of molecular parameters (photo-physics, enzymatic activity, binding affinity) Cellular and molecular biology studies (cell signaling, membrane dynamics) Ultra-sensitive chemical analysis (drug screening, medical diagnostics)

3 Jablonski Scheme of Fluorescence Photobleaching S1 Excitation T1 Fluorescence Emission S0

4 Main challenge of single molecule detection: Raman and Rayleigh scattering High-efficient optical filters Minimizing detection volume Long wavelength dyes Background ~ V Background ~ λ-4

5 Absoprtion Spectra of Standard Dyes and Autofluorescent Biomolecules Furan η Coumarine Fluorescein Rhodamine Oxazine Cyanine fl Thyrosin Coproporphyrine / Protoporphyrine Tryptophan Elastin Chlorophyll Collagen Flavins Wavelength (nm) Courtesy: Christoph Zander Uni GH Siegen

6 Fluorescence Correlation Spectroscopy

7 Confocal Fluorescence Microscopy

8 Principle of Confocal Detection Objective Dichroic mirror Tube lens Confocal aperture Towards detector

9 Fluorescence Intensity Fluctuations

10 Fluorescence Intensity Fluctuations: Autocorrelation

11 Fluorescence Intensity Fluctuations: Autocorrelation

12 Fluorescence Intensity Fluctuations: Autocorrelation

13 Structure of an autocorrelation curve

14 Example: Measured FCS curves of yellow fluorescent protein

15 Amplitude of an autocorrelation curve

16 Normalized amplitude of an autocorrelation curve

17 Ideal molecule detection function Molecule detection function (1/e2 isosurface) NA = 1.2 wd = 3 mm tubelens = 180 mm n0 = 1.33 λex = 635 nm ω = 4.9 mm focus pos. = 10 µm λem = 670 nm magn. = 60 pinhole radius = 50 µm

18 Cover-slide thickness deviation

19 Refractive index mismatch

20 Optical saturation

21 Intensity dependence of FCS (Alexa633) 30 µw 100 µw 300 µw 1 autocorrelation [a.u.] time [s]

22 Pulsed versus cw-excitation (Alexa633) 2.4 x 10-6 pulsed 635 nm cw 647 nm 2 2 apparent diffusion [cm /s] cw excitation power [µw]

23 Laser beam width and detection volume

24 2-focus confocal system

25 Time-tagged time-resolved mode of photon counting Frequency Fluorescence decay curve Data: t1 Laser pulse Decay time (ns) 15 t2 t3 t4 t5 t6 t7 t8 τ1 τ2 τ3 τ4 τ5 τ6 τ7 τ8

26 PIE: Pulsed interleaved excitation A Photon counts [a.u.] B A 0 5 B Time [ns] 20 25

27 Absolute FCS: two mutually shifted detection volumes

28 2fFCS of Atto655 in GdHCl: refractive index dependence

29 2fFCS: optical saturation dependence

30 Hard application of 2fFCS: 2+ Ca -binding of Calmodulin

31 2+ Ca -binding of Calmodulin: Hydrodynamic radius

32 Protein folding/unfolding: Tryptophan cage

33 Measuring fast conformational fluctuations of biomolecules Time scale of interest: nanoseconds up to milliseconds Probes: Förster resonance energy transfer Electron transfer Reporter: (i) Intensity (ii) Lifetime

34 Tryptophan induced fluorescence quenching of dye Atto I0/I τ/τ0 10 N 1.0 N O Trp [mm] O OH 60 N

35 Conformational dynamics of small peptide k+ k hν hν k0 k+ k k0

36 Conformational dynamics of small peptide (binding epitope of p53-antibody) 1 k + = 120 ns 1 k = 267 ns Pexc = 4 mw Pexc = 400 µw

37 Time-tagged time-resolved mode of photon counting Frequency Fluorescence decay curve Data: t1 Laser pulse Decay time (ns) 15 t2 t3 t4 t5 t6 t7 t8 τ1 τ2 τ3 τ4 τ5 τ6 τ7 τ8

38 FLCS Fluorescence lifetime correlation spectroscopy

39 FLCS Fluorescence lifetime correlation spectroscopy

40 FLCS: Working principle

41 FLCS Fluorescence lifetime correlation spectroscopy

42 Bi-exponential lifetime of a Cy5-streptavidin conjugate

43 FLCS of Cy5-Streptavidin

44 FLCS of Cy5-Streptavidin

45 FLCS of Cy5-Streptavidin 1.2 µ s τ = 1.7 ns τ = 0.7 ns A > 90 % A < 10 % 0.91 µ s 0.23 µ s 0.23 µ s 3.5 µ s 3.5 µ s 1.2 µ s dark state dark state 0.91 µ s

46 Single Molecule Imaging

47 Fluorescing molecule as an electric dipole Negative charge Amplitude Orientation Positive charge

48 The electric dipole: Near field, far field, and virtual photons Oscillating dipole is surrounded by virtual photons that are damped with increasing distance from the dipole. During return to the ground state, a propagating photons is emitted carrying away the excited state energy.

49 Angular distribution of emission Angular distribution of emitted radiation is given by the classical sin2θ law. In the quantum mechanical picture, the classical angular distribution of radiation corresponds to a probability of emitting a photon into a given direction.

50 Tunneling of evanescent modes into optically denser medium: Vertical dipole case upper medium n1 = 1.33 lower medium n2 = 1.33

51 Tunneling of evanescent modes into optically denser medium: Vertical dipole case

52 Emission into glass from a fluorescent molecule crossing a water/glass interface

53 Lifetime of fluorescent molecule crossing a water/glass interface

54 Collection efficiency of oil immersion microscope objective

55 Angular distribution of single molecules on glass surface

56 Defocused imaging of single molecules Microscope Table Oil Immersion 1.4 NA, 100 x PiFoc Dichroic Mirror Emission Filter Excitation/ Polarization Filter Tube Lens CCD KrAr nm

57 Theoretically calculated patterns

58 Defocused imaging of single molecules: pattern matching

59 Emission dipole hopping in a perylene tetrachromophore

60 Emission dipole hopping in a perylene tetrachromophore

61 Rotational diffusion of molecules Measurement: Hiroshi Uji-i

62 Rotational diffusion of molecules

63 Symmetric top Brownian rotator = D D cos Θ t = cos φ cos ψ sin φ sin ψ cos θ π 2π 2π t = d θ d φ d ψ sin θg ( φ, θ, ψ, t ) ( cos φ cos ψ sin φ sin ψ cos θ ) =e cos 2 Θ = t ( 2 D + ) t D t 1 ( 6 D + 4 ) t + e + e D + t 3 12 D + t 1 12 D +9 t = e( ) + e( ) + e( ) t D t 3 ( 6 D + 4 ) t 1 ( 20 D + 4 ) t 1 ( 20 D +16 ) t cos 4 Θ = + e 6 D t + e + e + e + e t cos3 Θ

64 Rotational diffusion of molecules: Correlation analysis D << D

65 Motor proteins: myosin V along actin

66 Myosin V moving along actin filament 1.45 oil immersion objective 160 x magnification 10 ms exposure time / frame defocusing 500 nm Measurement by Erdal Toprak, UIUC

67 Myosin motion and reorientation

68 Myosin motion and reorientation

69 Myosin motion and reorientation N = 97 molecules 1151 tilting events

70 Myosin motion and reorientation We observe that there is a consistent fluctuation of β between two well defined angles as myosin V steps. This is consistent with the lever arm hypothesis. Unlike β, the change in α shows no consistent or recognizable pattern which is an evidence for diffusional binding of myosin V.

71 Superresolution microscopy: Overcoming Abbe's resolution limit Fluorophore distribution (bar = 1µm) Confocal Laser Scanning Microscope (CLSM) (A tribute to microscopy pioneer Antoni van Leeuwenhoek)

72 intensity Spatial resolution limit of standard light microscopy position [µm]

73 Lateral resolution limit of standard light microscopy: Abbe's equation λ 2n.sinθ θ objective N.A. = n.sinθ

74 Laser Scanning Confocal Microscopy (LSCM) LSCM with deconvolution is completely equivalent in resolution power and photon usage with structured illumination microscopy laser beam objective PSF

75 Axial resolution limit of standard light microscopy e ik0 z +e k z,θ = θ ik z,θ z 2 = 2 + 2cos ( k0 k z,θ ) z n cos θ λ n k0 = λ objective λ n ( 1 cos θ )

76 4π microscopy standing wave generation by counter-propagating focusing of two coherent laser beams λ λ = n ( 1 cos θ4 π ) 2n laser beam 1st objective PSF 2nd objective laser beam

77 Back to basics: Physics of fluorescence Photobleaching S1 Excitation T1 Fluorescence Emission S0

78 Ground state depletion microscopy: Using saturation of the excited state

79 Stimulated Emission S1 STE Excitation S0 Fluorescence Emission

80 Stimulated Emission Depletion Microscopy excitation laser PSF STED laser

81 Stimulated Emission Depletion Microscopy

82 Stimulated Emission Depletion Microscopy

83 Temporal behavior of ground state depletion after sudden switch-on of excitation

84 µ s µ s 3.2 µ s 6.4 µ s x [µ m] rel. amplitude 0.4 µ s 320 nm 0.2 µ s 160 nm nm 0.1 µ s 40 nm 1 0 nm 0.0 µ s rel. amplitude Converting temporal into spatial information: Dynamic Saturation Optical Microscopy time [µ s] 8 10

85 Potential realization of Dynamic Saturation Optical Microscopy:

86 Potential realization of Dynamic Saturation Optical Microscopy:

87 Dynamic Saturation Optical Microscopy: Point spread function

88 Theoretical estimate of DSOM performance Fluorophore distribution (bar = 1µm) DSOM Confocal Laser Scanning Microscope (CLSM) DSOM + Bessel beam

89 Complex photophysics of Alexa647 Alexa 647

90 Combining DSOM and FCS Alexa 647

91 Ground state depletion into triplet state S1 T1 Excitation Fluorescence Emission S0 f ( r) = a ( r) 1 + τa ( r ) τkisc f ( r ) s ( r, t ) = + exp k ph + τkisc f ( r ) t k ph + τkisc f ( r ) k ph + τkisc f ( r ) k ph { }

92 Ground state depletion into metastable state (switchable chromophores) S1 Excitation M Fluorescence Emission S0 s ( r, t ) = exp { τktrans f ( r ) t} f ( r) = a ( r) 1 + τa ( r )

93 Ground state depletion into first excited state S1 Excitation Fluorescence Emission S0 s ( r, t ) = a ( r) { { 1 1 exp τ + a ( r ) t 1 τ + a( r) }}

94 Summary of DSOM Relatively simple: one laser only employing a standard CLSM pure electronic data evaluation relatively robust against aberration can be combined with 4π or other techniques Drawback: resolution enhancement limited to ca. 5 times

95 Publications available at

96 Acknowledgements/Cooperations Ingo Gregor Digambara Patra Jan Sykora Luru Dai Thomas Dertinger Iris von der Hocht Jörg Fitter Thomas Gensch Benjamin Kaupp (FZ Jülich) Markus Sauer (Univ. Bielefeld) Hiroshi Uji-i, Johan Hofkens (Katholieke Universiteit Leuven) Erdal Toprak, Paul Selvin (Univ. Illinois Urbana-Champaign)