Rainer Heintzmann

physik.uni.jena.de

Rainer Heintzmann

Hochaufl

Hochauflösende

Hochaufl

Hochaufl

Hochaufl sende sende

sende

sende

Fluorezenzmikroskopie basierend basierend

basierend

basierend auf

auf

auf

auf

computergest

computergestützter

computergest

computergest

computergest tzter tzter

tzter

Datenauswertung

Datenauswertung

Datenauswertung

Datenauswertung

Datenauswertung

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

Rainer Heintzmann, 2007


5

APD1

Leica 4Pi

APD2

http://www.leica-microsystems.com

Rainer Heintzmann, 2007


6

Leica 4Pi

http://www.leica-microsystems.com

Rainer Heintzmann, 2007


7

4Pi images

Deviding Escherichia Coli

From: Bahlmann, K., S. Jakobs

and S. W. Hell

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

Rainer Heintzmann, 2007


8

4Pi images

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

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

Rainer Heintzmann, 2007


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

Rainer Heintzmann, 2007


10

I5M M images and OTF

Mats Gustafsson,

UCSF, San Francisco

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

k x,y

k z

Rainer Heintzmann, 2007


11

Stimulated

Stimulated

Emission mission

Depletion epletion

Rainer Heintzmann, 2007


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

Rainer Heintzmann, 2007


Saturation of the stimulated emission

… provides the nonlinearity

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.

13

Rainer Heintzmann, 2007


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

Rainer Heintzmann, 2007


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

Rainer Heintzmann, 2007


17

STED Microscopy

STED beam Resulting PSF

1 µm

NA 1.3, 10nm pixelsize, no background

Rainer Heintzmann, 2007


18

STED Microscopy

STED beam Resulting PSF

1 µm

NA 1.3, 10nm pixelsize, 10% background,

STED PSF gets quite dim

Rainer Heintzmann, 2007


19

4Pi-STED 4Pi STED images

Fluorescent plane:

Rainer Heintzmann, 2007


20

STED Images Confocal STED

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.

Rainer Heintzmann, 2007


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

Rainer Heintzmann, 2007


22

STED Images

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

STED microscopy resolves nanoparticle assemblies.

New J. Phys. 8: 106.

Rainer Heintzmann, 2007


23

Computational spectacles

Methods requiring computation

Rainer Heintzmann, 2007


Paradigm: Optimize for direct visibility

+ →

Optics

Object Image

E.g.: Widefield, Confocal, STED

Does not necessarily optimize information content!

24

Rainer Heintzmann, 2007


Paradigm: Optimize for information content

Object

25

+ →

Optics

Data

Data Computation

Image

+ →

Rainer Heintzmann, 2007


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

Rainer Heintzmann, 2007


27

Overview

�� Pointilism: Localisation not resolution

�� Axial Tomography

�� Structured Illumination Microscopy

�� Circumventing the limit

… using Non-linear Non linear Effects

Rainer Heintzmann, 2007


28

Overview

�� Pointilism: Localisation not resolution

�� Axial Tomography

�� Structured Illumination Microscopy

�� Circumventing the limit

… using Non-linear Non linear Effects

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?

Rainer Heintzmann, 2007


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.

Rainer Heintzmann, 2007


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

10 frames of the times series.

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)

Rainer Heintzmann, 2007


34

Single Dot

10 frames of the times series.

Sum of time series ICA Results

ICA analysis finds only 1 source

Rainer Heintzmann, 2007


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

Rainer Heintzmann, 2007


36

Photo Activated Localization Microscopy

See also

STORM, FPALM

E. Betzig et al.,

Imaging Intracellular Fluorescent

Proteins at Nanometer Resolution

Science 313,

1642 - 1645

Rainer Heintzmann, 2007


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

Rainer Heintzmann, 2007


38

Overview

�� Pointilism: Localisation not resolution

�� Axial Tomography

�� Structured Illumination Microscopy

�� Circumventing the limit

… using Non-linear Non linear Effects

Rainer Heintzmann


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

Rainer Heintzmann


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.

Rainer Heintzmann


41

OTF in Axial Tomography

Aperture increase

by rotation of

the specimen

Rainer Heintzmann


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

Rainer Heintzmann


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 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

Rainer Heintzmann


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

Rainer Heintzmann


45

Biological Specimen

Polytrichum Commune

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

Moss Spore

Fully automatically

registered

Rainer Heintzmann


46

Biological Specimen

Polytrichum Commune

Moss Spore

Fully automatically

registered

Rainer Heintzmann


Selective elective Plane lane Illumination

llumination Microscopy

icroscopy

47

Plane of focus

Illumination

Unnessesary

Bleaching

Rainer Heintzmann


Selective elective Plane lane Illumination

llumination Microscopy

icroscopy

48

Illumination

Cylinder Lens

Light Sheet

Rainer Heintzmann


Selective elective Plane lane Illumination

llumination Microscopy

icroscopy

49

Illumination

Detection

Rainer Heintzmann


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)

Rainer Heintzmann


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

Rainer Heintzmann


52

Overview

�� Pointilism: Localisation not resolution

�� Axial Tomography

�� Structured Illumination Microscopy

�� Circumventing the limit

… using Non-linear Non linear Effects

Rainer Heintzmann, 2007


53

Linear

Structured Illumination

Rainer Heintzmann, 2007


54

Moiré Demo

Rainer Heintzmann


55

Patterned Excitation 2D Setup

lightsource

Rainer Heintzmann, 2007


56

I em (x) = Obj(x) · I ex (x)

Multiplication in real space


Convolution in Fourier space

~ ~ ~

Iem (k) = Obj(k) ⊗ Iex (k)

Rainer Heintzmann, 2007


57

Convolution

Rainer Heintzmann, 2007


58

Convolution

Rainer Heintzmann, 2007


59

Convolution

Rainer Heintzmann, 2007


60

Influence of optical imaging

…reconstructing the data?

Rainer Heintzmann, 2007


61

Separating the orders…

Rainer Heintzmann, 2007


62

Separating the orders…

Shifting the orders…

Rainer Heintzmann, 2007


63

Separating the orders…

Shifting the orders…

Recombining the orders…

Rainer Heintzmann, 2007


64

Separating the orders…

Shifting the orders…

Recombining the orders… using the correct weights.

Rainer Heintzmann, 2007


65

Sample Widefield Image Reconstructed Image

Rainer Heintzmann, 2007


66

Experimental Setup

Rainer Heintzmann, 2007


Structured Illumination experiments

67

Rainer Heintzmann, 2007


Structured Illumination experiments

68

Rainer Heintzmann, 2007


Structured Illumination experiments

69

Grating

Resolution Enhanced ~2×

Rainer Heintzmann, 2007


4 Directions: A Puzzle

70

Rainer Heintzmann, 2007


Structured Illumination experiments

71

2 µm

Rainer Heintzmann, 2007


Structured Illumination experiments

72

Sample: Darren Williams, Guy Tear

Drosophila

Polyteen Squash

Sitox Green

Rainer Heintzmann, 2007


Structured Illumination experiments

73

Sample: Darren Williams, Guy Tear

Drosophila

Polytene Squash

Sitox Green

Rainer Heintzmann, 2007


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.

Rainer Heintzmann, 2007


75

Overview

�� Pointilism: Localisation not resolution

�� Axial Tomography

�� Structured Illumination Microscopy

�� Circumventing the limit

… using Non-linear Non linear Effects

Rainer Heintzmann, 2007


Circumventing

1

0

relative

transfer

|kx,y| x,y|

[1/m]

the Abbe Resolution Limit

requires nonlinearity

76

Rainer Heintzmann, 2007


77

Non-linear

Non linear

Structured Illumination

Rainer Heintzmann, 2007


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

Rainer Heintzmann, 2007


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

Rainer Heintzmann, 2007


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)

Rainer Heintzmann, 2007


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

Rainer Heintzmann, 2007


The Abbe limit has been

circumvented,

the new limit is a matter of

signal and noise

82

Rainer Heintzmann, 2007


Saturation

of

Photoswitching

as non-linear non linear effect

83

Rainer Heintzmann, 2007


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

Rainer Heintzmann, 2007


85

Intensity

Photoswitchable Proteins

Object: 2 Pairs of lines

dark

state

Low intensity activation

(~20% activated)

position

bright

state

depletion light

(= read light)

Rainer Heintzmann, 2007


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

Rainer Heintzmann, 2007


87

Conclusion:

Computational spectacles

can help to see the nature

in greater detail

Rainer Heintzmann, 2007


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, 2007

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