Microscopy beyond the diffraction limit
Bierwagen et.al. Nano Lett. 2010
Introduction to subdiffractional Microscopy
Dr. Jakob Bierwagen – Université de Genève Department Chimie Physique 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
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 Microscope
200 nm
xz-View xy-View of a confocal PSF of a confocal PSF
x 500 nm 2nsin Ernst Abbe 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 1IOFF ISat Activation 0 Excitation S1 S0 rSMM S 2nsin N 0 switch-on of single isolated molecules