Rainer Heintzmann

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Rainer Heintzmann, 

Heintzmann 

Rainer.Heintzmann@kcl.ac.uk 

Randall Division, King‘s King College London 

RANDALL 

division of cell and 

molecular biophysics 

Jena, November 2007

Overview: Direct Imaging 

�� 4Pi Microscopy, Scanning Wavefield + I 5M �� STED Microscopy

3 

Bigger NA: 

4Pi Microscopy 

Rainer Heintzmann, 2007

4 

Aperture increase: 4 Pi Microscope (Type C) 

Dichromatic 

Beamsplitter 

Sample between 

Coverslips 

Detector 

Pinhole 

Fluorescence Emisssion 

2 Photon Effect 

z 

Laser 

Stefan W. Hell, 

Max Planck Inst. bpc, 

Goettingen, Germany 

High 

Sidelobes 

Fluorescence 

Intensity 

z 


5 

APD1 

Leica 4Pi 

APD2 

http://www.leica-microsystems.com 


6 

Leica 4Pi 

http://www.leica-microsystems.com 


7 

4Pi images 

Deviding Escherichia Coli 

From: Bahlmann, K., S. Jakobs 

and S. W. Hell 

(2001). Ultramicr. 87: 155-164. 


8 

4Pi images 

Confocal (2-Photon ) 4Pi (2-Photon) 

Thanks to: Elisabeth Ehler, Reiner Rygiel, Martin Fiala, Tanjef Szellas 


9 

Aperture increase (3): ( ): 

I5 Microscope 

icroscope 

incoherent 

ncoherent illumination 

llumination interference 

nterference 

imag mage interference 

nterference 

Sample 

CCD 

Camera 

z 

Dichroic Mirror 

White Light 

Source 

Offset!! 

Fluorescence Emisssion 

• No XY scanning required 

(full field illum., full field det.) 

• No laser required 

• Higher background 

no pinhole ⇒ more signal 

Fluorescence 

Intensity 

z 


10 

I5M M images and OTF 

Mats Gustafsson, 

UCSF, San Francisco 

http://www.msg.ucsf.edu/sedat/mats/ 

k x,y 

k z 


11 

Stimulated 

Stimulated 

Emission mission 

Depletion epletion 


12 

STED Microscopy 

Klar, T. A., S. Jakobs, M. Dyba, A. Egner and S. W. Hell 

(2000). Proc.Nat. Acad. Sc. U.S.A. 97(15): 8206-8210. 

P s1 

~P s0 σI ex 

P s0 

Singlet 

ps 

ps 

Stimulated 

Emission 

Depletion 


Saturation of the stimulated emission 

… provides the nonlinearity 



13 


STED, the details 

14 

Intensity 

First 

(excitation) 

Dispersion Element 

Or Pulsed Laser Diode! 

STED, the details 

15 

Intensity 

Excitation 

Emission 

wavelength 

554nm, 250fs 

Detection Range 

STED 

745-760nm, 13ps 

Low efficiency 

⇒ Very strong light 


STED, selecting a dye 

16 

S 2 

Singlet 

S 1 

~P s0σI ex 

S 0 

ps 

ps 

~µs 

∼P s1 τ –1 ~ I ex 

Triplet 

T 1 

T 0 


17 


STED beam Resulting PSF 

1 µm 

NA 1.3, 10nm pixelsize, no background 


18 


STED beam Resulting PSF 

1 µm 

NA 1.3, 10nm pixelsize, 10% background, 

STED PSF gets quite dim 


19 

4Pi-STED 4Pi STED images 

Fluorescent plane: 


20 

STED Images Confocal STED 




21 

STED Images 

Confocal 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 


22 

STED Images 

Willig, K. I., J. Keller, M. Bossi, S. W. Hell (2006): 

STED microscopy resolves nanoparticle assemblies. 

New J. Phys. 8: 106. 


23 

Computational spectacles 

Methods requiring computation 


Paradigm: Optimize for direct visibility 

+ → 

Optics 

Object Image 

E.g.: Widefield, Confocal, STED 

Does not necessarily optimize information content! 

24 


Paradigm: Optimize for information content 

Object 

25 

+ → 

Optics 

Data 

Data Computation 

Image 

+ → 


26 

Examples in Medical Imaging 

MRI 

http://www.cis.rit.edu/htbooks/mri/images/head.gif 

PET 

http://www.cerebromente.org.br/n01/pet/petdep.gif 

CT 

http://www.vetmed.lsu.edu/vth&c/Orthopedics/Images/ 

Computed%20Tomography%20(CT)%20Scanner.RV.jpg 

SPECT 

http://www.physics.ubc.ca/research/images/spect.gif 

fMRI 

http://www.fmri.wfubmc.edu/other%20pics/ 

lab_brain_logo.JPG 


27 

Overview 

�� Pointilism: Localisation not resolution 

�� Axial Tomography 

�� Structured Illumination Microscopy 

�� Circumventing the limit 

… using Non-linear Non linear Effects 


28 

Overview 






Rainer Heintzmann

29 

Localization not resolution 

Localization not resolution 

If positions are know you can 

paint a picture! 

If particles can be separated, 

their relative positions can be 

measured accurately 

Rainer Heintzmann, Seurat: 2007 Tiger

30 

How to separate particles? 

Spectral precision distance microscopy 

Problems: Chromatic Aberrations, few dyes 

P. Edelmann, A. Esa, H. Bornfleth, R..Heintzmann, M. Hausmann, and C. Cremer. Proc. of SPIE , 3568:89-95, 1999 

Using fluorescence lifetime for separation (FLIM) 

Problems: Lifetime depends on microenvironm. 

M. Heilemann, D.P. Herten, R.Heintzmann, C. Cremer, C. Müller, P. Tinnefeld, K.D. Weston, 

J. Wolfrum and M. Sauer. Anal. Chem., 74, 3511-3517, 2002. 

Can we use the blinking characteristics? 


31 

Can we separate Quantum Dots by their individual blinking? 

�� Localization >10 times 

better than resolution 

�� Quantum dots blink 

slow enough for imaging 

�� QDs blink independent 

�� Can we separate, 

even with strong overlap? 655 nm quantum dots 

on a cover slip 

K.A. Lidke, B. Rieger, T.M. Jovin, R. Heintzmann Optics Express 13, 7052-7062, 2005. 


Large Separation 

10 frames of the times series. 

Sum of time series 

ICA analysis finds 2 blinking sources 

Separation 535 nm 

ICA Results 

32 K.A. Lidke, B. Rieger, T.M. Jovin, R. Heintzmann Optics Express 13, 7052-7062, Rainer 2005. 

Heintzmann, 2007

33 

Close Separation 


Sum of time series ICA Results 

ICA analysis identifies 2 closely spaced blinking sources 

Can resolve 2 spots at 23 nm. (current max: 5 spots) 


34 

Single Dot 


Sum of time series ICA Results 

ICA analysis finds only 1 source 


Outlook 

•Process entire images (many particles) 

Resolution by in Microscopy 

•Use of specific QD-blinking statistical distribution 

•Use of spatial information 

•Use temporal information 

K.A. Lidke, B. Rieger, T.M. Jovin, R. Heintzmann Optics Express 13, 7052-7062, 2005. 

35 


36 

Photo Activated Localization Microscopy 

See also 

STORM, FPALM 

E. Betzig et al., 

Imaging Intracellular Fluorescent 

Proteins at Nanometer Resolution 

Science 313, 

1642 - 1645 


37 

Photo Activated Localization Microscopy 

WF PALM 

EM 

E. Betzig et al., Science 313, 1642 – 1645 (2006) 

Mitochondria 

COS-7 cells 

cryo sections Cytochrom C oxidase import sequence - dEosFP 


38 

Overview 







39 

Problem: Limited Numerical 

umerical Aperture perture 

z 

x,y 

Cell 

Objective Lense 

Immersion Medium 

Cover Slip 

Slide 

α 

Immersion 

Medium 

NA = n sin α 


40 

Solution: Axial Tomography 

z 

Cell 

x,y 

Objective Lense 

Immersion Medium 

Cover Slip 

Glass Fiber 

Immersion 

Medium 

Aperture increase 

by rotation of 

the specimen 

Shaw et al., 

Cogswell et al., 

Kawata et al., 

Heintzmann et al. 


41 

OTF in Axial Tomography 

Aperture increase 

by rotation of 

the specimen 


42 

Axial Tomography Experiments 

Confocal image (1 view) of quartz beads 

∅ 416 nm 

RITC 

Quartz 

∅ 200 nm 

R. Heintzmann and C. Cremer., J. Microsc., 206 (1), 7-23, 2002 

R. Heintzmann, G. Kreth, and C. Cremer, Analytical Cellular Pathology, 20(1):7-15, 2000 


43 

Reconstructed Image 

Cluster of quartz beads 

Positions of the quartz beads 

∅ 416 nm 

RITC 

Quartz 

∅ 200 nm 

One bead as PSF, 

Segmentation of Result 


R. Heintzmann, G. Kreth, and C. Cremer, Analytical Cellular Pathology, 20(1):7-15, 2000 


44 

Combined ML-Deconvolution 

ML Deconvolution 

View 1 View 2 

Measured 

Register 

View 1 View 2 

M i 

Reconstructed Estimate 

Apply 

, 

Back 

Convolution 

Convolution 

Compare 

Simulated 

, 

C NC 

E i 


45 

Biological Specimen 

Polytrichum Commune 


Moss Spore 

Fully automatically 

registered 


46 

Biological Specimen 

Polytrichum Commune 

Moss Spore 

Fully automatically 

registered 


Selective elective Plane lane Illumination 

llumination Microscopy 

icroscopy 

47 

Plane of focus 

Illumination 

Unnessesary 

Bleaching 




icroscopy 

48 

Illumination 

Cylinder Lens 

Light Sheet 




icroscopy 

49 

Illumination 

Detection 


50 

Results (SPIM) 

View 1 Deconv. Combined Deconv. 

c. elegans embryo, i-lineage cells expressing RAP11-GFP (GTPase) 

as endocytotic marker 

Data: MPI Dresden, Sebastian Höpfner, Marino Zerial 

(collaboration with Zeiss) 


Electrodes 

51 

Results (Cell Suspensions) 

Cell in Suspension 

View 1, Deconvolved: 

View 2, Deconvolved: 

Combined Deconvolution: 

Data: Olivier Renaud, Spencer Shorte (Inst. Pasteur), 

SW13/20, lamin-GFP (green), mito-tracker (red), 

spinning disc system (Andor) 

5 µm 

O. Renaud, R. Heintzmann, A. Sáez-Cirión, T. Schnelle, T. Muller and S. Shorte, Proc. SPIE, 

6441, 10.1117/12.699000, 2007 


52 

Overview 







53 

Linear 

Structured Illumination 


54 

Moiré Demo 


55 

Patterned Excitation 2D Setup 

lightsource 


56 

I em (x) = Obj(x) · I ex (x) 

Multiplication in real space 

↕ 

Convolution in Fourier space 

~ ~ ~ 

Iem (k) = Obj(k) ⊗ Iex (k) 


57 

Convolution 


58 

Convolution 


59 

Convolution 


60 

Influence of optical imaging 

…reconstructing the data? 


61 

Separating the orders… 


62 


Shifting the orders… 


63 



Recombining the orders… 


64 



Recombining the orders… using the correct weights. 


65 

Sample Widefield Image Reconstructed Image 


66 

Experimental Setup 


Structured Illumination experiments 

67 



68 



69 

Grating 

Resolution Enhanced ~2× 


4 Directions: A Puzzle 

70 



71 

2 µm 



72 

Sample: Darren Williams, Guy Tear 

Drosophila 

Polyteen Squash 

Sitox Green 



73 

Sample: Darren Williams, Guy Tear 

Drosophila 

Polytene Squash 

Sitox Green 


74 

Application in Ophtalmology? 

Ophtalmology 

Structured illumination, 

a method for higher resolution and 

better optical sectioning in retinal 

imaging ? 

Local radiation density can be kept low. 


75 

Overview 







Circumventing 

1 

0 

relative 

transfer 

|kx,y| x,y| 

[1/m] 

the Abbe Resolution Limit 

requires nonlinearity 

76 


77 

Non-linear 

Non linear 

Structured Illumination 


78 

Two Spaces 

Linear Excitation (low intensity) 

magnitude 

Border of 

detection OTF 

-ϕ0 ϕ ~ 

0 Obj +1 (k) 

-K 0 0 K0 Non-Linear Excitation (high 

intensity) 

magnitude 

Border of 

detection OTF 

-3ϕ 0 

-3K 0 

-2ϕ 0 

-2K 0 

-ϕ0 

-K 0 

0 

ϕ 0 

K 0 

spatial 

frequency 

2ϕ0~ Obj +2 (k) 

spatial 

frequency 

R. Heintzmann, T.M. Jovin, and C. Cremer., J. Opt. Soc. Am. A,19 (8), 1599-1609, 2002 


R. Heintzmann, T.M. Jovin, and C. Cremer., 

J. Opt. Soc. Am. A,19 (8), 1599-1609, 2002 

79 

PSF comparison 

SPEM (7x7) 

70 nm 

λ em = 520 nm 

SPEM (9x9) 

PEM 

118 nm Widefield 

57 nm 215 nm 


80 

1 µm 

2D Patterned SPEM Approach 

Conventional 

microscopy 

Mats Gustafsson, U.C. San Francisco: 

1 µm 

50 nm microspheres 

Linear 

structured 

illumination 

M.G.L. Gustafsson (2005), PNAS, 37, 13081-13086 

1 µm 

Saturated 

structured 

illumination 

(3 new harmonics, 

53 J/m 2 , triangle apodized) 


81 

2D Patterned SPEM Approach 

Line profile through bead 

FWHM 

Mats Gustafsson, U.C. San Francisco: 

– including the bead diameter of ~51 nm. 

Distribution of FWHM 

50 60 70 (nm) 

Mean FWHM = 58.6 nm 

Estimate FWHM of PSF: 59 2 - (51/ 2 ) 2 = 46 nm 

M.G.L. Gustafsson (2005), PNAS, 37, 13081-13086 


The Abbe limit has been 

circumvented, 

the new limit is a matter of 

signal and noise 

82 


Saturation 

of 

Photoswitching 

as non-linear non linear effect 

83 


84 

Photoswitchable Proteins 

DRONPA 

(photoswitchable protein) 

for resolution improvement 

- Transfectable, living cells 

- Low power lighsources 

Dron: to hide 

Pa: to appear 

See also: 

Anemonia Sulcata 

purple fluorescent protein 

asFP595, 

Diheteroarylethenes 


85 

Intensity 

Photoswitchable Proteins 

Object: 2 Pairs of lines 

dark 

state 

Low intensity activation 

(~20% activated) 

position 

bright 

state 

depletion light 

(= read light) 


86 

Intensity 

Moving the pattern 

Object: 2 Pairs of lines 

dark 

state 

position 

bright 

state 

Expected Resolution: < 50 nm 

depletion light 

(= read light) 

Related to 

SPEM + STED 


87 

Conclusion: 

Computational spectacles 

can help to see the nature 

in greater detail 


88 

Acknowledgement 

�� Liisa Hirvonen, Kai Wicker, 

Ondrej Mandula, 

Yannick Colpin, 

Mats Gustafsson, 

Christoph Cremer (PEM, Axial) 

Elisabeth Ehler, Darren Williams (PEM) 

Gustafsson, (PEM, SPEM) 

�� Thomas Jovin, Quentin Hanley, 

Keith Lidke, Bernd Rieger (PAM, Pointillism)

Rainer Heintzmann

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