Hybrid Single and Dual Pattern Structured Light Illumination
STED etc Wicker - uni-jena.deOptical... · microscopy Saturated structured illumination 1 µm...
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2/15/2013 1 Advanced Optical Microscopy lecture 17. December 2012 Kai Wicker Today: Nonlinear fluorescence microscopy: Going beyond the Abbe limit ‐ Non‐uniform illumination: doubling the Abbe limit ‐ Non‐linear sample response, effective illumination ‐ Multi‐photon microscopy ‐ Stimulated emission depletion (STED) ‐ Non‐linear structured illumination (nl‐SIM) Non‐uniform illumination: doubling the Abbe limit
Transcript of STED etc Wicker - uni-jena.deOptical... · microscopy Saturated structured illumination 1 µm...
2/15/2013
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Advanced Optical Microscopylecture
17. December 2012Kai Wicker
Today:
Nonlinear fluorescence microscopy:Going beyond the Abbe limit
‐ Non‐uniform illumination: doubling the Abbe limit‐ Non‐linear sample response, effective illumination‐Multi‐photon microscopy‐ Stimulated emission depletion (STED)‐ Non‐linear structured illumination (nl‐SIM)
Non‐uniform illumination:
doubling the Abbe limit
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Wide‐field microscope
Perfect image: ⊗Fourier image:
Diffraction limited
Confocal microscope
Confocal PSF:
Confocal OTF: ⊗
Structured illumination
Raw image: ⊗SIM OTF: | | ⊗
Diffraction limited
Diffraction limited
Using non‐uniform illumination, the Abbe limit can be extended by about a factor 2!
Confocal microscope
Confocal PSF:
Confocal OTF: ⊗
Structured illumination
Raw image: ⊗SIM OTF: | | ⊗
Wide‐field
Confocal 0.3AU
Non‐linear sample response,
effective illumination
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Illumination intensity
Emitted fluorescence
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Fluorescence saturation
Diffraction limit
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Diffraction limit
We illuminate with a light distribution of intensity .The sample responds, as if illuminated with .
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Non‐linear response to diffraction limited illumination=
Linear response to non‐diffraction limited (effective) illumination
Confocal microscope
Confocal PSF:
Confocal OTF: ⊗
Structured illumination
Raw image: ⊗SIM OTF: | | ⊗
NOT diffraction limited
NOT diffraction limited
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Examples of non‐linear sample responses in fluorescence
‐ Fluorescence saturation used in SIM, confocal
‐ Multi‐photon absorption used in two‐, three‐photon microscopy
‐ Photo‐switching of fluorophores used in SIM, localisation microscopy
‐ Stimulated emission used in STED microscopy
Multi‐photon microscopy
Jablonski diagram
Absorption…
… and spontaneous emission
Normal fluorescence
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Jablonski diagram
NO absorption…
Normal fluorescence
Jablonski diagram
2‐photon absorption…
… and spontaneous emission
2‐photon fluorescence
2‐photon fluorescence
‐ 2‐photon absorption requires two photons to be present simultaneously
‐ The probability for this grows quadratically with intensity
‐ It will only occur where the local intensity is high
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Probability of a photon being at a certain position :
In Fourier space:
Probability of two photons being at a certain position :
In Fourier space: ⊗
Probability of photons being at a certain position :
In Fourier space: [ ⊗ …⊗
However: For N‐photon absorption, an N‐times smaller wavelength is needed! no gain !
Stimulated emission depletion (STED)
Jablonski diagram
Absorption…
… and spontaneous emission
Spontaneous emission
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Jablonski diagram
Absorption…
… and stimulated emission
Stimulated emission
Excitation beamSTED beam
ExcitationIntensity of stimulated emission beamRemaining excitation after stimulated emission
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ExcitationIntensity of stimulated emission beamRemaining excitation after stimulated emission
ExcitationProbability of stimulated emission (i.e. switching off)Remaining excitation after stimulated emission
Resolution of STED
Conventional Abbe limit
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STED limit
2 1
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The STED principleThe phase mask
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phase mask
phase mask in back focal plane
554nm, 250fs STED745‐760nm, 13ps
Low efficiency Very strong light
wavelength
Excitation
Detection range
Emission
Intensity
The STED principle
The STED setup
Image: Busko et al., Micron 43(5), 563‐588 (2012)
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STED ImagesConfocal STED
human embryonic kidney labeled with a red-emitting dye (MR 121SE)Microtubules Immunofluorescence
Current Opinion in Biotechnology 2005, 16:3–12
From micro to nano: recent advances in high-resolution microscopy; Yuval Garini, Bart J Vermolen and Ian T Young
STED Images
STED beam
excitation
Hell 2008, Nature Methods, 6,24‐32
Vimentin
STED Images
Resolution down to 8 nm (N+Vacancy in Diamond)
STED microscopy reveals crystal colour centres with nanometric resolution, E. Rittweger, K. Y. Han, S. E. Irvine, C. Eggeling and S. W. Hell, Nature Photonics 2009, DOI: 10.1038/NPHOTON.2009.2
Current STED state of the art ‐ resolution
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Non‐linear structured illumination (nl‐SIM)
Real space
Fourier space Diffraction limit
Saturated SIM
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magnitude
spatial frequency
Support regionof OTF
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magnitude
spatial frequency
‐K0 K0‐2K0
‐K0 K0
Linear Excitation (low intensity)
Non‐Linear Excitation (high intensity)
‐3K0
Support regionof OTF
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Conventionalmicroscopy
Saturatedstructured illumination
1 µm
Linearstructured illumination
1 µm1 µm
Mats Gustafsson, UCSF50 nm microscpheresnonlinearity: fluorescence saturation, 53J/m2
3 extra harmonics
M.G.L. Gustafsson (2005), PNAS, 37, 13081‐13086
NonlinearStructured Illumination Micropscopy
Real space
Fourier space Diffraction limit
Saturated SIM
Problem 1:Very high total intensity
Problem 2:Low energy in high frequencies
Non‐linear structured illumination (nl‐SIM)
Using photo‐switchable fluorophores
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Photo‐switchable fluorophores can be switched between an “on”‐state (dark) and an “off”‐state (bright.
photo‐switchable fluorophores
E.g.:Activation: 405nmFluorescence excitation + deactivation: 488nm
Image: http://zeiss‐campus.magnet.fsu.edu/tutorials/superresolution/resolft/index.html
„Switching‐off“ lightProbability of fluorophores being in „on“‐state
Nuclear pores of a human embryonic kidney(Rego et al. (2012), PNAS, 109, E135‐E143)
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End of lecture
