Nanoscopy with Focused Light
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1 Nanoscopy with Focused Light Stefan W. Hell Max Planck Institute for Biophysical Chemistry Department of NanoBiophotonics Göttingen & German Cancer Research Center (DKFZ) Optical Nanoscopy Division Heidelberg Nobel Lecture in Chemistry, Stockholms universitet 8. December 2014
2 200 nm ½ wavelength of light
3 Light microscopy: most popular microscopy technique in life sciences Electron microscopy, etc. 20 % 80 % Light microscopy
4 Fluorescent labels indicate biomolecule of interest S 1 Excitation S 0
5 200 nm ½ wavelength of light
6 because of the diffraction barrier: d 2nsin 500 nm 200 nm Wavelength Lens Verdet (1869) Abbe (1873) Helmholtz (1874) Rayleigh (1874)
7 because of the diffraction barrier: d 2nsin Photomultiplier or APD 500 nm 200 nm Lens Verdet (1869) Abbe (1873) Helmholtz (1874) Rayleigh (1874)
8 because of the diffraction barrier: d 2nsin Eye 500 nm 200 nm Lens Verdet (1869) Abbe (1873) Helmholtz (1874) Rayleigh (1874)
9 because of the diffraction barrier: d 2nsin Camera 500 nm 200 nm Lens Verdet (1869) Abbe (1873) Helmholtz (1874) Rayleigh (1874)
10 Standard (Confocal) STED Jena, Germany
11 What I believed around 1990: the resolution limiting effect of diffraction can be overcome ( ) by fully exploiting the properties of the fluorophores. Combined with modern quantum optical techniques the scanning (confocal) microscope has the potential of dramatically improving the resolution in far-field light microscopy. SWH, Opt. Commun. 106 (1994) accepted November 1993
12 What I believed around 1990: the resolution limiting effect of diffraction can be overcome ( ) by fully exploiting the properties of the fluorophores. Combined with modern quantum optical techniques the scanning (confocal) microscope has the potential of dramatically improving the resolution in far-field light microscopy.
13 d 2nsin Lens Son 1 Keep molecules in a dark state! stimulated emission S 0 dark
14 d 2nsin Lens S 1 Keep molecules in a dark state! S 0 dark
15 d 2nsin Lens Son 1 Keep molecules in a dark state! stimulated emission S 0 dark
16 Fluor. ability STED microscope: Hell & Wichmann, Opt. Lett. (1994) Lens Sample x 200 nm y PhaseMod 2p 0 d excitation (ON) Laser de-excitation (OFF) d λ 2n sin on S 1 fluorescence t fl ~ns 1.0 (S on 1 ) (S 0 ) excitation stimulated emission 0.5 S 0 t vib < 1ps 0 I s [GW/cm²]
17 Fluor. ability STED microscope: Hell & Wichmann, Opt. Lett. (1994) Lens Sample x 200 nm y PhaseMod 2p 0 d ON Laser OFF d λ 2n sin on S 1 fluorescence t fl ~ns 1.0 (S on 1 ) (S 0 ) excitation stimulated emission 0.5 S 0 t vib < 1ps 0 I s [GW/cm²]
18 Fluor. ability STED microscope: Hell & Wichmann, Opt. Lett. (1994) Lens Sample x 200 nm y PhaseMod 2p 0 d ON Laser OFF on S 1 fluorescence t fl ~ns 1.0 (S on 1 ) (S 0 ) excitation stimulated emission 0.5 S 0 t vib < 1ps 0 I s [GW/cm²]
19 Fluor. ability STED microscope: Hell & Wichmann, Opt. Lett. (1994) Lens Sample x 200 nm y PhaseMod 2p 0 ON Laser OFF on S 1 fluorescence t fl ~ns 1.0 (S on 1 ) (S 0 ) excitation stimulated emission 0.5 S 0 t vib < 1ps 0 I s I [GW/cm²]
20 Fluor. ability STED microscope: Hell & Wichmann, Opt. Lett. (1994) Lens Sample x 200 nm y PhaseMod 2p 0 ON Laser OFF on S 1 fluorescence t fl ~ns 1.0 (S on 1 ) (S 0 ) excitation stimulated emission 0.5 S 0 t vib < 1ps 0 I s I [GW/cm²]
21 Fluor. ability STED microscope: Hell & Wichmann, Opt. Lett. (1994) Lens Sample x 200 nm y PhaseMod 2p 0 ON Laser OFF on S 1 fluorescence t fl ~ns 1.0 (S on 1 ) (S 0 ) excitation stimulated emission 0.5 S 0 t vib < 1ps 0 I s I [GW/cm²]
22 Fluor. ability STED microscope: Hell & Wichmann, Opt. Lett. (1994) Lens Sample x 200 nm y PhaseMod 2p 0 ON Laser OFF on S 1 fluorescence t fl ~ns 1.0 (S on 1 ) (S 0 ) excitation stimulated emission 0.5 S 0 t vib < 1ps 0 I s I [GW/cm²]
23 Fluor. ability STED microscope: Hell & Wichmann, Opt. Lett. (1994) Lens Sample x 200 nm y PhaseMod 2p 0 ON Laser OFF on S 1 fluorescence t fl ~ns 1.0 (S on 1 ) (S 0 ) excitation stimulated emission 0.5 S 0 t vib < 1ps 0 I s I [GW/cm²]
24 Fluor. ability STED microscope: Hell & Wichmann, Opt. Lett. (1994) Lens Sample x 200 nm y PhaseMod 2p 0 ON Laser OFF on S 1 fluorescence t fl ~ns 1.0 (S on 1 ) (S 0 ) excitation stimulated emission 0.5 S 0 t vib < 1ps 0 I s I [GW/cm²]
25 Fluor. ability STED microscope: Hell & Wichmann, Opt. Lett. (1994) Lens Sample x 200 nm y PhaseMod 2p 0 ON Laser OFF on S 1 fluorescence t fl ~ns 1.0 (S on 1 ) (S 0 ) excitation stimulated emission 0.5 S 0 t vib < 1ps 0 I s I [GW/cm²]
26 Fluor. ability STED microscope: Hell & Wichmann, Opt. Lett. (1994) Lens Sample x 200 nm y PhaseMod 2p 0 ON Laser OFF d λ 2n sin on S 1 fluorescence t fl ~ns 1.0 (S on 1 ) (S 0 ) excitation stimulated emission 0.5 S 0 t vib < 1ps 0 I s I [GW/cm²]
27 Fluor. ability STED microscope: Hell & Wichmann, Opt. Lett. (1994) Lens Sample x 200 nm y PhaseMod 2p 0 ON Laser OFF on S 1 fluorescence t fl ~ns 1.0 (S on 1 ) (S 0 ) excitation stimulated emission 0.5 S 0 t vib < 1ps 0 I s I [GW/cm²]
28 Fluor. ability STED microscope: Hell & Wichmann, Opt. Lett. (1994) Lens Sample x 200 nm y PhaseMod 2p 0 ON Laser OFF on S 1 fluorescence t fl ~ns 1.0 (S on 1 ) (S 0 ) excitation stimulated emission 0.5 S 0 t vib < 1ps 0 I s I [GW/cm²]
29 Fluor. ability STED microscope: Hell & Wichmann, Opt. Lett. (1994) Lens Sample x 200 nm y PhaseMod 2p 0 ON Laser OFF on S 1 fluorescence t fl ~ns 1.0 (S on 1 ) (S 0 ) excitation stimulated emission 0.5 S 0 t vib < 1ps 0 I s I [GW/cm²]
30 Fluor. ability STED microscope: Hell & Wichmann, Opt. Lett. (1994) Lens Sample x 200 nm y PhaseMod 2p 0 ON Laser OFF on S 1 fluorescence t fl ~ns 1.0 (S on 1 ) (S 0 ) excitation stimulated emission 0.5 S 0 t vib < 1ps 0 I s I [GW/cm²]
31 Protein assemblies in cell Standard (Confocal) Nuclear pore complex 250nm Göttfert, Wurm et al Biophys J (2013)
32 Protein assemblies in cell STED Nuclear pore complex 250nm Göttfert, Wurm et al Biophys J (2013)
33 Protein assemblies in cell STED 150nm Nuclear pore complex 250nm Göttfert, Wurm et al Biophys J (2013)
34 Viral infection Env HIV Envelope protein on single virions immature mature Confocal STED Confocal STED STED STED Insight: Env proteins are assembled in mature HIV 300nm 100nm HIV (Vpr.eGFP) Env (Ab 2G12) 100nm J Chojnacki,..,SWH, HG Kräusslich, Science (2012)
35 Synaptic vesicles in axon of living hippocampal neuron Standard (Confocal) snapshot Synaptotagmin immunostained Scale: 300 nm Westphal, Rizzoli, Lauterbach, Jahn, SWH, Science (2008)
36 Synaptic vesicles in axon of living hippocampal neuron Video rate STED Synaptotagmin immunostained 28 frames/ second Scale: 300 nm Westphal, Rizzoli, Lauterbach, Jahn, SWH, Science (2008)
37 Neurophysiology STED YFP in living mouse brain 23 x 18 x 3 µm, 10µs / px, 800 x 600 x 5 px, interval 5 min ~20 µm deep 2 µm Cortical neurons expressing cytoplasmic EYFP 1 µm Berning et al, Science (2012)
38 The resolution
39 Fluorescence STED microscope: x 200 nm Lens Sample y PhaseMod 2p 0 d ON Laser OFF d λ 2n sin on S 1 fluorescence t fl ~ns 1.0 (S on 1 ) (S 0 ) excitation stimulated emission 0.5 S 0 t vib < 1ps 0 I s I [GW/cm²]
40 Fluorescence STED microscope: x 200 nm Lens Sample y PhaseMod 2p 0 d ON Laser OFF d λ 2n sin on S 1 fluorescence t fl ~ns 1.0 (S on 1 ) (S 0 ) excitation stimulated emission 0.5 S 0 t vib < 1ps 0 I s I [GW/cm²]
41 Fluorescence STED microscope: x 200 nm Lens Sample y PhaseMod 2p 0 d ON Laser OFF d λ 2n sin on S 1 fluorescence t fl ~ns 1.0 (S on 1 ) (S 0 ) excitation stimulated emission 0.5 S 0 t vib < 1ps 0 I s I [GW/cm²]
42 Fluorescence STED microscope: x 200 nm Lens Sample y PhaseMod 2p 0 d ON Laser OFF d λ 2n sin on S 1 fluorescence t fl ~ns 1.0 (S on 1 ) (S 0 ) excitation stimulated emission 0.5 S 0 t vib < 1ps 0 I s I [GW/cm²]
43 Fluorescence STED microscope: x 200 nm Lens Sample y PhaseMod 2p 0 d ON Laser OFF d λ 2n sin 1 I I s on S 1 fluorescence t fl ~ns 1.0 (S on 1 ) (S 0 ) excitation stimulated emission 0.5 S 0 t vib < 1ps 0 I s I [GW/cm²]
44 Fluorescence STED microscope: x 200 nm Lens Sample y PhaseMod 2p 0 d ON Laser OFF d λ 2n sin 1 I I s 0 on S 1 fluorescence t fl ~ns 1.0 (S on 1 ) (S 0 ) excitation stimulated emission 0.5 S 0 t vib < 1ps 0 I s I [GW/cm²]
45 Fluorescence STED microscope: x 200 nm Lens Sample y PhaseMod 2p 0 d ON Laser OFF d λ 2n sin 1 I I s 0 on S 1 fluorescence t fl ~ns 1.0 (S on 1 ) (S 0 ) excitation stimulated emission 0.5 S 0 t vib < 1ps 0 I s I [GW/cm²]
46 Fluorescence STED microscope: x 200 nm Lens Sample y PhaseMod 2p 0 d ON Laser OFF d λ 2n sin 1 I I s 0 on S 1 fluorescence t fl ~ns 1.0 (S on 1 ) (S 0 ) excitation stimulated emission 0.5 S 0 t vib < 1ps 0 I s I [GW/cm²]
47 Fluorescence STED microscope: x 200 nm Lens Sample y PhaseMod 2p 0 d ON Laser OFF d λ 2n sin 1 I I s 0 on S 1 fluorescence t fl ~ns 1.0 (S on 1 ) (S 0 ) excitation stimulated emission 0.5 S 0 t vib < 1ps 0 I s I [GW/cm²]
48 Material sciences, magnetic sensing, quantum information x 200 nm y NV - in diamond V d N Color centers 0 (1-dimensional) 2.4 nm 4.2 nm = 775 nm
49 Principle: Discern by ON / OFF states in the sample STED GSD (metastable dark state) RESOLFT on S 1 fluoresc. t ~ ns S 1 on fluoresc. T 1 t ~ ms on cis trans S 0 Hell, Wichmann, Opt Lett (1994) S 0 Hell, Kroug Appl Phys B (1995) Hell, Nat Biotech (2003) t ~ ms d λ 2n sin 1 I I s 1.0 on ~ (S 1/t 0 ) I s 2 4 6
50 Principle: Discern by ON / OFF states in the sample STED GSD (metastable dark state) RESOLFT on S 1 fluoresc. t ~ ns MW/cm 2 S 1 on fluoresc. T 1 t ~ ms kw/cm 2 on cis trans S 0 Hell, Wichmann, Opt Lett (1994) S 0 Hell, Kroug Appl Phys B (1995) Hell, Nat Biotech (2003) W/cm 2 t ~ ms d λ 2n sin 1 I I s 1.0 on ~ (S 1/t 0 ) I s 2 4 6
51 RESOLFT: many doughnuts (zeros) in parallel λ 2n sin on because of low intensity operation.
52 RESOLFT: many doughnuts (zeros) in parallel on because of low intensity operation.
53 n pixels RESOLFT: many doughnuts (zeros) in parallel n pixels on because of low intensity operation.
54 RESOLFT Keratin filaments in living kidney epithelial cells recorded with >100,000 doughnuts in 2 seconds on Chmyrov et al, Nature Meth (2013) Scale bar: 10 µm
55 What does it take to get the best resolution?
56 20 th century: separate features by focusing light Good lenses! Object
57 20 th century: separate features by focusing light Object
58 Solution: separate by molecular (on/) states Object on S 1 S 1 on S 0 S 0 T 1 on cis trans etc.
59 STED, GSD, SSIM, RESOLFT, separate by molecular (on/) states on S 1 S 1 on S 0 S 0 T 1 on cis trans etc.
60 STED, GSD, SSIM, RESOLFT, separate by molecular (on/) states X,Y,Z controlled by incident light pattern on S 1 S 1 on S 0 S 0 T 1 on cis trans etc.
61 STED, GSD, SSIM, RESOLFT, separate by molecular (on/) states X,Y,Z controlled by incident light pattern on S 1 S 1 on S 0 S 0 T 1 on cis trans etc.
62 STED, GSD, SSIM, RESOLFT, controlled by incident light pattern PALM, STORM, PAINT, GSDIM, X,Y,Z Betzig et al (2006) Rust et al (2006) Hess et al (2006) Moerner et al (1989) Orrit et al (1990) single molecule!
63 STED, GSD, SSIM, RESOLFT, controlled by incident light pattern PALM, STORM, PAINT, GSDIM, X,Y,Z? stochastic Betzig et al (2006) Rust et al (2006) Hess et al (2006) Moerner et al (1989) Orrit et al (1990) single molecule!
64 STED, GSD, SSIM, RESOLFT, controlled by incident light pattern Camera PALM, STORM, PAINT, GSDIM, X,Y,Z x,y,z? stochastic centroid of emitted light pattern Betzig et al (2006) Rust et al (2006) Hess et al (2006) Moerner et al (1989) Orrit et al (1990) single molecule!
65 STED, GSD, SSIM, RESOLFT, ~ 1 I I s Camera and many photons for X,Y,Z precision X,Y,Z x,y,z? stochastic ~ 1 n Betzig et al (2006) Rust et al (2006) Hess et al (2006) Moerner et al (1989) Orrit et al (1990) PALM, STORM, PAINT, GSDIM, single molecule!
66 STED, GSD, SSIM, RESOLFT, on separates the features Camera Betzig et al (2006) Rust et al (2006) Hess et al (2006) Moerner et al (1989) Orrit et al (1990) PALM, STORM, PAINT, GSDIM, single molecule!
67 Superresolution separates features using (at least) 2 molecular states on fluorescent non-fluorescent
68 Superresolution separates features using (at least) 2 molecular states on A B fluorescent absorbing scattering spin up non-fluorescent non-absorbing non-scattering spin down
69 Superresolution separates features using (at least) 2 molecular states on A B fluorescent absorbing scattering spin up non-fluorescent non-absorbing non-scattering spin down
70 since 1997 since 2003 Former lab members Current lab members Collaborators: P. Hänninen, E. Soini, R. Jahn, F. Barrantes, S. Sigrist, H.G. Kräusslich, S. Rizzoli
71 0 down to molecular scale. Standard STED ON OFF
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