beyond the diffraction limit

Bierwagen et.al. Nano Lett. 2010

Introduction to subdiffractional Microscopy

Dr. Jakob Bierwagen – Université de Genève Department Chimie Physique – The process Jablonski Diagram

O O OH

OH

O

O OH Fluoresceine Fluorescence – The process Jablonski Diagram

O O OH

OH

O

O OH Fluoresceine Fluorescence – The process Jablonski Diagram

O O OH

OH

O

O OH Fluoresceine Fluorescence – The process Jablonski Diagram

tvib≈ 1ps

tfl≈ 2-4ns

tvib≈ 1ps

15000 O O OH

10000 OH

O

5000 Photons per bin Photons O OH

0 Fluoresceine 0 5 10 15 20 25 Time in ns Confocal

200 nm

xz-View xy-View of a confocal PSF of a confocal PSF

 x  500 nm 2nsin  1873 200 nm Confocal Microscope

200 nm

xz-View xy-View of a confocal PSF of a confocal PSF

 x  500 nm 2nsin  Ernst Abbe 1873 200 nm Switching the markers

488 nm 594 nm ~50 ps ~100 ps

Fluorescence ~ns Pulsed Time/ns excitation

STED (STimulated Emission Depletion) Switching the markers

488 nm 594 nm ~50 ps ~100 ps

Fluorescence ~ns Pulsed Time/ns excitation

1.0 Challenges: correct timing

ON and wavelength 0.5 (S1) OFF (S ) STED (STimulated Emission Depletion) Fluorescence 0

0 2 4 6

2 ISTED [GW/cm ] The Phasemask The Phasemask The Phasemask The Phasemask From a confocal to a STED- microscope

z

x Detector 200 nm

y

S0 S1

S0 excitation S0

200 nm

Pulsed

S 1 t fl 1sn fluorescence

Excitation

t vib 1ps S0 From a confocal to a STED- microscope

z

x Detector 200 nm

y

S0 S1

S0 excitation Depletion S0 (STED) Sync 200 nm

Pulsed Pulsed 1.0 S 1 t fl 1sn ON fluorescence (S1) 0.5 stimulated Emission Excitation OFF

(S0) Fluorescenc

t vib 1ps 0 S0 2 4 6 2 ISTED [GW/cm ] From a confocal to a STED- microscope

z

x Detector 200 nm

y PhaseMod 2p 0 S0 S1

S0 excitation Depletion S0 (STED) Sync 200 nm

Pulsed Pulsed 1.0 S 1 t fl 1sn ON fluorescence (S1) 0.5 stimulated Emission Excitation OFF

(S0) Fluorescenc

t vib 1ps 0 S0 2 4 6 2 ISTED [GW/cm ] From a confocal to a STED- microscope

z

x Detector 200 nm

y PhaseMod 2p 0 S0 S1

S0 excitation Depletion S0 (STED) Sync 200 nm

Pulsed Pulsed 1.0 S 1 t fl 1sn ON fluorescence (S1) 0.5 stimulated Emission Excitation OFF

(S0) Fluorescenc

t vib 1ps 0 S0 2 4 6 2 ISTED [GW/cm ] STED-Microscopy dynamic resolution scaling

continuosly scalable resolution in the nanometer region

20nm Crimson beads 633nm exc, 90ps, 30kW/cm2 785nm STED 200ps, 76MHz Resolution scaling with STED

1

220 nm

0 1.0 -200 0 200

0.5

Fluorescence

0.0 0 100 200 300 Power [mW]

Harke et al., Opt. Exp., 2008 Resolution scaling with STED

1

132 nm

0 1.0 -200 0 200

0.5

Fluorescence

0.0 0 100 200 300 Power [mW]

Harke et al., Opt. Exp., 2008 Resolution scaling with STED

1

84 nm

0 1.0 -200 0 200

0.5

Fluorescence

0.0 0 100 200 300 Power [mW]

Harke et al., Opt. Exp., 2008 Resolution scaling with STED

1

52 nm

0 1.0 -200 0 200

0.5

Fluorescence

0.0 0 100 200 300 Power [mW]

Harke et al., Opt. Exp., 2008 Resolution scaling with STED

1

22 nm

0 1.0 -200 0 200

0.5

Fluorescence

0.0 0 100 200 300 Power [mW]

Harke et al., Opt. Exp., 2008 Derivation of the modified Abbe equation ON

tvib≈ 1ps

tfl≈ 2-4ns STED-Pulse <100ps

tvib≈ 1ps OFF

V. Westphal et.al., Phys. Rev. Lett. 2005 Derivation of the modified Abbe equation

Δ푟푠푡푒푑 = FWHM = 2*r (@ heff = 1/2)

Approximation = exc ≈ sted Resolution of STED and practical limitations

Bleaching Bleaching Modified Abbe equation  r  2nsin 1 I STED / I sat Bleaching

Bleaching

Rankin, Biophys. J., 2011

Wildanger et al. Opt. Exp. 2009, Leutenegger et al. Opt. Exp. 2010 Hotta et al., JACS, 2010 , Wildanger et al., J. Microsc., 2009 Resolution of STED and practical limitations

Bleaching Bleaching Modified Abbe equation  r  2nsin 1 I STED / I sat Bleaching

Bleaching

Rankin, Biophys. J., 2011

Wildanger et al. Opt. Exp. 2009, Leutenegger et al. Opt. Exp. 2010 Hotta et al., JACS, 2010 , Wildanger et al., J. Microsc., 2009 Time gated detection – increasing contrast and resolution

15000 Excitation

10000

5000

Photons perPhotons bin Fluorescence

0 0 5 10 15 20 25 Time in ns

Vicidomini, Nature Methods, 2011 Time gated detection – increasing contrast and resolution

15000

15000 STED-Light Excitation 10000 10000

5000 5000

Photons perPhotons bin Fluorescence Photons perPhotons bin 0 0 5 10 15 20 25 0 Time in ns 0 5 10 15 20 25 Time in ns

Vicidomini, Nature Methods, 2011 Time gated detection – increasing contrast and resolution

15000

15000 STED-Light Excitation 10000 10000

5000 5000

Photons perPhotons bin Fluorescence Photons per bin Photons

0 Detection-window 0 5 10 15 20 25 0 Time in ns 0 5 10 15 20 25 Time in ns

Vicidomini, Nature Methods, 2011 Time gated detection – increasing contrast and resolution

15000

15000 STED-Light Excitation 10000 10000

5000 5000

Photons perPhotons bin Fluorescence Photons per bin Photons

0 Detection-window 0 5 10 15 20 25 0 Time in ns 0 5 10 15 20 25 Time in ns

Vicidomini, Nature Methods, 2011 Switching

ON Conditions: • Reversible switchable tvib≈ 1ps mechanism • Saturable by light OFF ON tfl≈ 1ns STED-Pulse

tvib≈ 1ps OFF

Modified Abbe equation  r  2nsin 1 I STED / I sat

1 퐼푠푎푡∝ 휏푓푙 Fluorescent Proteins

Ser65 Tyr66 Gly67 OH O

R NH R 1 NH NH 2 O O

Aequorea victoria OH (http://mabryonline.org)

avGFP Maturation - 1961 discovered by Osamura

Shimomura O O R - ~26 kDa 2 - 11-stranded b-sheet N - autocatalytical formation of N HO the chromophor NH HO R 1 Reversible swichtable red fluorescent proteins switching of rsCherryRev (negativ switching)

on-switching 430nm Advantages of red proteins: • low absorption of cells in the red region off-switching 592nm => low phototoxicity • reduced scattering =>deep-tissue imaging possible • multicolour imaging

Applications: high resolution microscopy data storage

Stiel et.al. Biophys. J., 2008 Breaking the diffraction barrier with red fluorescent reversible switchable Proteins

Lavoie-Cardinal, Bierwagen et.al. ChemPhysChem, 2014 RESOLFT Microsocpy - parallelisation

Chmyrov et al., Nat. Meth. 2013

Parallelisation => fast acquisition of large images Method of wide field high resolution microscopy Central Principle: Stochastic Switching and Readout

Realization with PALM and STORM:

Activation Camera Switch Excitation image off switch-on of single isolated molecules

 rloc  2nsin N Adding the frames Switching into Dark states Cis - trans (Cyan-Dyes/Proteins)

Singlet – Triplet Betzig et al. Science 2006 Rust et al. Nat Meth. 2006 Using Conventional Dyes – GSDIM Microscopy

Nmean≈858 Photons/event rSMS ≈ 10-12 nm Real: 25 nm

Fölling et al. Nat Meth. 2008 Bierwagen et. al. Nano Lett. 2010 Hell, Patent DE1020060213 17B3, 2006 Similarities and differences between RESOLFT and single-molecule Microscopy RESOLFT Single molecule Microscopy

• Many photons for switching • Very few photons for switching • Very few photons for detecting • Many photons for detecting => Precision by confining the => Precision by Localizing the fluorescence volume center of fluorescence Switching is the central mechanism • Mathematical resolution • All optical resolution improvements improvements • Targeted switching • Random switching • Optically more challenging Summary - high resolution microscopy

Conditions: Detector • Reversible switchable mechanism PhaseMod • Saturable by light 2p 0

OFF ON Excitation Depletion (STED)

x 200 nm  1 y xRESOLFT   S 2NA 1IOFF ISat Activation 0 Excitation S1  S0 rSMM  S 2nsin N 0 switch-on of single isolated molecules