Nanoscopy with Focused Light Stefan W. Hell Max Planck Institute - PowerPoint PPT Presentation

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

200 nm ½ wavelength of light

Light microscopy: most popular microscopy technique in life sciences Electron microscopy, etc. 20 % 80 % Light microscopy

Fluorescent labels indicate biomolecule of interest S 1 S 0 Excitation

200 nm ½ wavelength of light

… because of the diffraction barrier:  d   2 n sin 500 nm  200 nm  Wavelength Detector Lens Verdet (1869) Abbe (1873) Helmholtz (1874) Rayleigh (1874)

… because of the diffraction barrier:  d   2 n sin 500 nm Photomultiplier or APD  200 nm Detector Lens Verdet (1869) Abbe (1873) Helmholtz (1874) Rayleigh (1874)

… because of the diffraction barrier:  d   2 n sin 500 nm Eye  200 nm Detector Lens Verdet (1869) Abbe (1873) Helmholtz (1874) Rayleigh (1874)

… because of the diffraction barrier:  d   2 n sin 500 nm Camera  200 nm Detector Lens Verdet (1869) Abbe (1873) Helmholtz (1874) Rayleigh (1874)

STED Standard (Confocal) Jena, Germany

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

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.”

 d   2 n sin  Lens S 1 on S 1 Keep molecules in a dark state ! stimulated emission dark S 0 off

 d   2 n sin  Lens S 1 S 1 Keep molecules in a dark state ! dark S 0

 d   2 n sin  Lens S 1 on S 1 Keep molecules in a dark state ! stimulated emission dark S 0 off

STED microscope: Hell & Wichmann, Opt. Lett. (1994) Lens x Sample 200 nm y Detector PhaseMod d 2 p 0 de-excitation excitation (ON) (OFF) Laser λ  d  2 n sin 1.0 t fl ~ n s on S 1 (S 0 ) (S 1 ) on off Fluor. ability fluorescence 0.5 excitation stimulated emission t vib < 1ps S 0 off 0 I s [GW/cm²] 2 4 6

STED microscope: Hell & Wichmann, Opt. Lett. (1994) Lens x Sample 200 nm y Detector PhaseMod d 2 p 0 ON OFF Laser λ  d  2 n sin 1.0 t fl ~ n s S 1 on (S 0 ) (S 1 ) on on off off Fluor. ability fluorescence 0.5 excitation stimulated emission t vib < 1ps S 0 off 0 I s [GW/cm²] 2 4 6

STED microscope: Hell & Wichmann, Opt. Lett. (1994) Lens x Sample 200 nm y Detector PhaseMod d 2 p 0 ON OFF Laser 1.0 t fl ~ n s S 1 on (S 0 ) (S 1 ) on off Fluor. ability fluorescence 0.5 excitation stimulated emission t vib < 1ps S 0 off 0 I s [GW/cm²] 2 4 6

STED microscope: Hell & Wichmann, Opt. Lett. (1994) Lens x Sample 200 nm y Detector PhaseMod 2 p 0 ON OFF Laser 1.0 t fl ~ n s S 1 on (S 0 ) (S 1 ) on off Fluor. ability fluorescence 0.5 excitation stimulated emission t vib < 1ps S 0 off 0 I s I [GW/cm²] 2 4 6

STED microscope: Hell & Wichmann, Opt. Lett. (1994) Lens x Sample 200 nm y Detector PhaseMod 2 p 0 ON OFF Laser 1.0 t fl ~ n s S 1 on (S 0 ) (S 1 ) on off Fluor. ability fluorescence 0.5 excitation stimulated emission t vib < 1ps S 0 off 0 I s I [GW/cm²] 2 4 6

STED microscope: Hell & Wichmann, Opt. Lett. (1994) Lens x Sample 200 nm y Detector PhaseMod 2 p 0 ON OFF Laser 1.0 t fl ~ n s S 1 on (S 0 ) (S 1 ) on off Fluor. ability fluorescence 0.5 excitation stimulated emission t vib < 1ps S 0 off 0 I s I [GW/cm²] 2 4 6

STED microscope: Hell & Wichmann, Opt. Lett. (1994) Lens x Sample 200 nm y Detector PhaseMod 2 p 0 ON OFF Laser 1.0 t fl ~ n s S 1 on (S 0 ) (S 1 ) on off Fluor. ability fluorescence 0.5 excitation stimulated emission t vib < 1ps S 0 off 0 I s I [GW/cm²] 2 4 6

STED microscope: Hell & Wichmann, Opt. Lett. (1994) Lens x Sample 200 nm y Detector PhaseMod 2 p 0 ON OFF Laser 1.0 t fl ~ n s S 1 on (S 0 ) (S 1 ) on off Fluor. ability fluorescence 0.5 excitation stimulated emission t vib < 1ps S 0 off 0 I s I [GW/cm²] 2 4 6

STED microscope: Hell & Wichmann, Opt. Lett. (1994) Lens x Sample 200 nm y Detector PhaseMod 2 p 0 ON OFF Laser 1.0 t fl ~ n s S 1 on (S 0 ) (S 1 ) on off Fluor. ability fluorescence 0.5 excitation stimulated emission t vib < 1ps S 0 off 0 I s I [GW/cm²] 2 4 6

STED microscope: Hell & Wichmann, Opt. Lett. (1994) Lens x Sample 200 nm y Detector PhaseMod 2 p 0 ON OFF Laser 1.0 t fl ~ n s S 1 on (S 0 ) (S 1 ) on off Fluor. ability fluorescence 0.5 excitation stimulated emission t vib < 1ps S 0 off 0 I s I [GW/cm²] 2 4 6

STED microscope: Hell & Wichmann, Opt. Lett. (1994) Lens x Sample 200 nm y Detector PhaseMod 2 p 0 ON OFF Laser λ  d  2 n sin 1.0 t fl ~ n s S 1 on (S 0 ) (S 1 ) on off Fluor. ability fluorescence 0.5 excitation stimulated emission t vib < 1ps S 0 off 0 I s I [GW/cm²] 2 4 6

STED microscope: Hell & Wichmann, Opt. Lett. (1994) Lens x Sample 200 nm y Detector PhaseMod 2 p 0 ON OFF Laser 1.0 t fl ~ n s S 1 on (S 0 ) (S 1 ) on off Fluor. ability fluorescence 0.5 excitation stimulated emission t vib < 1ps S 0 off 0 I s I [GW/cm²] 2 4 6

STED microscope: Hell & Wichmann, Opt. Lett. (1994) Lens x Sample 200 nm y Detector PhaseMod 2 p 0 ON OFF Laser 1.0 t fl ~ n s S 1 on (S 0 ) (S 1 ) on off Fluor. ability fluorescence 0.5 excitation stimulated emission t vib < 1ps S 0 off 0 I s I [GW/cm²] 2 4 6

STED microscope: Hell & Wichmann, Opt. Lett. (1994) Lens x Sample 200 nm y Detector PhaseMod 2 p 0 ON OFF Laser 1.0 t fl ~ n s S 1 on (S 0 ) (S 1 ) on off Fluor. ability fluorescence 0.5 excitation stimulated emission t vib < 1ps S 0 off 0 I s I [GW/cm²] 2 4 6

STED microscope: Hell & Wichmann, Opt. Lett. (1994) Lens x Sample 200 nm y Detector PhaseMod 2 p 0 ON OFF Laser 1.0 t fl ~ n s S 1 on (S 0 ) (S 1 ) on off Fluor. ability fluorescence 0.5 excitation stimulated emission t vib < 1ps S 0 off 0 I s I [GW/cm²] 2 4 6

Protein assemblies in cell Standard (Confocal) Nuclear pore complex 250nm Göttfert, Wurm et al Biophys J (2013)

Protein assemblies in cell STED Nuclear pore complex 250nm Göttfert, Wurm et al Biophys J (2013)

Protein assemblies in cell STED 150nm 150nm Nuclear pore complex 250nm 1 1 8 8 2 2 7 7 3 3 6 6 4 4 5 5 Göttfert, Wurm et al Biophys J (2013)

Viral infection Env HIV Env elope protein on single virions immature mature Confocal STED STED Confocal STED STED 300nm Insight: Env proteins are assembled in mature HIV 100nm 100nm HIV (Vpr.eGFP) J Chojnacki,.., SWH , HG Kräusslich, Science (2012) Env (Ab 2G12)

Synaptic vesicles in axon of living hippocampal neuron Standard (Confocal) snapshot Synaptotagmin immunostained Scale: 300 nm Westphal, Rizzoli, Lauterbach, Jahn, SWH, Science (2008)

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) Westphal, Rizzoli, Lauterbach, Jahn, SWH, Science (2008)

Neurophysiology STED YFP in living mouse brain ~20 µm deep 23 x 18 x 3 µm, 10µs / px, 800 x 600 x 5 px, interval 5 min 2 µm Cortical neurons expressing cytoplasmic EYFP 1 µm Berning et al, Science (2012)

The resolution

STED microscope: x 200 nm Lens y Sample Detector d PhaseMod 2 p 0 ON OFF Laser λ d   2 n sin 1.0 t fl ~ n s S 1 on (S 1 ) on (S 0 ) off Fluorescence fluorescence stimulated emission 0.5 excitation t vib < 1ps S 0 off 0 I s I [GW/cm²] 2 4 6

STED microscope: x 200 nm Lens y Sample Detector d PhaseMod 2 p 0 ON OFF Laser λ d   2 n sin 1.0 t fl ~ n s S 1 on (S 1 ) on (S 0 ) off Fluorescence fluorescence stimulated emission 0.5 excitation t vib < 1ps S 0 off 0 I s I [GW/cm²] 2 4 6

STED microscope: x 200 nm Lens y Sample Detector d PhaseMod 2 p 0 ON OFF Laser λ d   2 n sin 1.0 t fl ~ n s S 1 on (S 1 ) on (S 0 ) off Fluorescence fluorescence stimulated emission 0.5 excitation t vib < 1ps S 0 off 0 I s I [GW/cm²] 2 4 6