Single Molecule Spectroscopy and Imaging

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

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)

Jablonski Scheme of Fluorescence Photobleaching S1 Excitation T1 Fluorescence Emission S0

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

Absoprtion Spectra of Standard Dyes and Autofluorescent Biomolecules Furan η Coumarine Fluorescein Rhodamine Oxazine Cyanine fl Thyrosin Coproporphyrine / Protoporphyrine Tryptophan Elastin Chlorophyll Collagen Flavins 300 400 500 Wavelength (nm) 600 700 Courtesy: Christoph Zander. 1999 Uni GH Siegen

Fluorescence Correlation Spectroscopy

Confocal Fluorescence Microscopy

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

Fluorescence Intensity Fluctuations

Fluorescence Intensity Fluctuations: Autocorrelation

Structure of an autocorrelation curve

Example: Measured FCS curves of yellow fluorescent protein

Amplitude of an autocorrelation curve

Normalized amplitude of an autocorrelation curve

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

Cover-slide thickness deviation

Refractive index mismatch

Optical saturation

Intensity dependence of FCS (Alexa633) 30 µw 100 µw 300 µw 1 autocorrelation [a.u.] 0.8 0.6 0.4 0.2 0-5 10 10-4 time [s] 10-3 10-2 10-1

Pulsed versus cw-excitation (Alexa633) 2.4 x 10-6 pulsed excitation @ 635 nm cw excitation @ 647 nm 2 2 apparent diffusion [cm /s] 2.2 1.8 1.6 1.4 1.2 1 0.8 0 200 400 600 cw excitation power [µw] 800 1000

Laser beam width and detection volume

2-focus confocal system www.microscopyu.com

Time-tagged time-resolved mode of photon counting Frequency Fluorescence decay curve Data: t1 Laser pulse -5 0 5 10 Decay time (ns) 15 t2 t3 t4 t5 t6 t7 t8 τ1 τ2 τ3 τ4 τ5 τ6 τ7 τ8

PIE: Pulsed interleaved excitation 1 3 5 7... 2 4 6 8... A Photon counts [a.u.] B A 0 5 B 10 15 Time [ns] 20 25

Absolute FCS: two mutually shifted detection volumes

2fFCS of Atto655 in GdHCl: refractive index dependence

2fFCS: optical saturation dependence

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

2+ Ca -binding of Calmodulin: Hydrodynamic radius

Protein folding/unfolding: Tryptophan cage

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

Tryptophan induced fluorescence quenching of dye Atto655 20 2.0 15 1.5 I0/I τ/τ0 10 N 1.0 N O 5 0 0 10 20 30 Trp [mm] 40 50 O OH 60 N

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

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

Time-tagged time-resolved mode of photon counting Frequency Fluorescence decay curve Data: t1 Laser pulse -5 0 5 10 Decay time (ns) 15 t2 t3 t4 t5 t6 t7 t8 τ1 τ2 τ3 τ4 τ5 τ6 τ7 τ8

FLCS Fluorescence lifetime correlation spectroscopy

FLCS: Working principle

FLCS Fluorescence lifetime correlation spectroscopy

Bi-exponential lifetime of a Cy5-streptavidin conjugate

FLCS of Cy5-Streptavidin

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

Single Molecule Imaging

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

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.

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.

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

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

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

Lifetime of fluorescent molecule crossing a water/glass interface

Collection efficiency of oil immersion microscope objective

Angular distribution of single molecules on glass surface

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 450-700 nm

Theoretically calculated patterns

Defocused imaging of single molecules: pattern matching

Emission dipole hopping in a perylene tetrachromophore

Rotational diffusion of molecules Measurement: Hiroshi Uji-i

Rotational diffusion of molecules

Symmetric top Brownian rotator = D D cos Θ t = cos φ cos ψ sin φ sin ψ cos θ π 2π 2π 0 0 0 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 1 1 6 D t 1 ( 6 D + 4 ) t + e + e 3 6 2 3 2 D + t 3 12 D + t 1 12 D +9 t = e( ) + e( ) + e( ) t 5 20 4 1 1 9 20 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 5 7 280 7 14 8 cos3 Θ

Rotational diffusion of molecules: Correlation analysis D << D

Motor proteins: myosin V along actin

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

Myosin motion and reorientation

Myosin motion and reorientation N = 97 molecules 1151 tilting events

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.

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

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

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

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

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 )

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

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

Ground state depletion microscopy: Using saturation of the excited state

Stimulated Emission S1 STE Excitation S0 Fluorescence Emission

Stimulated Emission Depletion Microscopy excitation laser PSF STED laser

Stimulated Emission Depletion Microscopy

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

0.8 0.7 0.6 0.5 0.8 µ s 0.4 0.3 1.6 µ s 3.2 µ s 6.4 µ s 0 0.1 0.2 x [µ m] 0.3 0.4 0.5 1 0.2 0.9 0.1 0.8 0 0.7 rel. amplitude 0.4 µ s 320 nm 0.2 µ s 160 nm 0.9 80 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 0.6 0.5 0.4 0.3 0.2 0.1 0 0 2 4 6 time [µ s] 8 10

Potential realization of Dynamic Saturation Optical Microscopy:

Dynamic Saturation Optical Microscopy: Point spread function

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

Complex photophysics of Alexa647 Alexa 647

Combining DSOM and FCS Alexa 647

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 { }

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 )

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) }}

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

Publications available at www.joerg-enderlein.de

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)