Start Pane Microscopie confocale - Télécom ParisTech€¦ · 4 Introduction Historique La microscopie photonique a été inventée au XVIIe siècle mais c'est à partir du XIXe

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Microscopie confocale

Start Pane

2

Introduction & Généralités

Principe de la microscopie confocale

Fluorescence – Sondes Fluorescentes et intérêts en biologie

Confocalité – avantages

Setup expérimental

Paramétrage d‘images

Applications

3D / Reconstruction d‘images

Quantifications

Macros

Deconvolution (PSF)

Limitations

Etudes de colocalisation

Calibration

Résolution

3

Généralités

Introduction




Setup expérimental


Applications


Quantifications

Macros

Deconvolution (PSF)

Limitations


Calibration

Résolution

4

Introduction

Historique

La microscopie photonique a été inventée au XVIIe siècle mais c'est à partir

du XIXe siècle qu'elle devient un outil indispensable pour les grandes

découvertes en biologie. La cellule découverte par Hooke en 1665 à l'aide d'un

microscope rudimentaire est précisée par Purkinje en 1825. La découverte du

noyau est faite par Brown en 1831. On découvre alors la division cellulaire et le

rôle du noyau. La microscopie photonique permet la découverte d'agents

pathogènes : le bacille de la lèpre par Hansen en 1874, de la tuberculose par

Koch en 1882, de la peste par Yersin en 1894.

Parallèlement à ces découvertes, les techniques de microscopie progressent. Le

premier microscope inversé date de 1850, et le premier condenseur permettant

d'optimiser l'éclairage est mis au point par Abbe en 1872, et optimisé par Köhler

en 1893.

La microscopie de fluorescence est imaginée par Reichert en 1908, puis

expérimentée avec des fluorochromes par Haitinger en 1911. Le contraste de

phase, décrit par Zernike date de 1934, et le contraste interférentiel est mis au

point par Nomarski en 1952.

5

World of small structures

Introduction

6

Cell Diagram, A small world of high complexity

Introduction

7

Introduction

8

ESSENTIALS

• Magnification : The way to see small detail

• Resolution : The way to distinguish between small detail

• Contrast : The way to see resolved and magnified detail

To see the Micro-cosmos

We need Microscopy

Introduction

9

• Human eye : 100000 nm

• Simple magnifier : 10000 nm

• Optical microscope : 200 nm limits

• Electron microscope : 0.5 – 3 nm

• Scanning Probe microscope : 0.1 – 10 nm

Microscopical range

Introduction

10

Imaging Modes

BF

PH

DIC

FL

Introduction

11

• Only specific structures are stained and images

• Unwanted structures remain are not visible

• Detail can be seen even if smaller than resolution limits

• With the advent of special dyes, staining of living cells is now possible

Contrast using Fluorescence

Introduction

12

Fluorescence

Jablonski Diagram

Energy

Prof. Alexander Jablonski, 1935

Absorption

Emission

Stokes-Shift

488nm

525nm

E = hn = hc/l

Excited Energy levels

Ground Energy levels

13

Prof. Alexander Jablonski, 1935

E = hn = hc/l

Principe - Fluorescence

14


15

Dichroic spectral position


16

Ultraviolets InfrarougesVisible

400 nm 700 nm

Sources lumineuses


17


18


19

Prof. Ploem’s invention


20


21


Sondes fluorescentes et intérêts en biologie

22

• sans modulation

dichroïque

• jusqu‘au 200 points

chaque spectre

• 2-5 nm résolution

xFP Spectra

0

0.2

0.4

0.6

0.8

1

400 450 500 550 600 650 700

wavelength (nm)

No

rmalized

CFP

BFP

eGFP

YFP

dsRed

Leica TCS SP2 AOBS: vrais spectres

In-situ Fluorescence protein spectraCourtesy Dr. T. Zimermann, EMBL, Heidelberg, Germany

23


Immunomarquages

Protéines de fusion

Autofluorescence

Sondes ioniques

Qdot nano-cristaux

Rendement quantique

Le rendement quantique de

fluorescence décrit l'efficacité

de fluorescence des molécules

après absorption de photons

SPECIFICITE DU SIGNAL

IMPORTANT :

STABILITE

24

Red Fluorescent Proteins

25

Red Fluorescent Proteins

Ref.: Campbell et al.

2002, PNAS, Vol. 99,

no.12: 7877-82

•DsRed

•mRFP1

26

Laser Orange 594 nm:

Nouveaux Fluorochrome, Excitation Optimal

Fluorochrome excité par 594 nm Excitation [nm] Emission [nm]

Alexa 568 577 603

5-ROX 578 604

HC Red 590 614

SNARF (carboxy) pH 9 576 639

Alexa 594 594 618

Bodipy TR-X SE 588 616

Calcium Crimson 588 611

CTC 5-cyano-2,3-ditolyl-Tetrazolium-Chloride 602 630

Red FluoSpheres 580 603

Texas Red 595 620

YO-PRO-3 613 629

27

Généralités

Introduction




Multiphoton

Setup expérimental


Applications


Quantifications

Macros

Deconvolution (PSF)

Limitations


Calibration

Résolution

28


Conventional vs Confocal

29

Résolution optimale Intensité de fluorescence

maximale


30

Basement membrane labeled with cy2 (green)Neurons labeled with cy3 (red)

Résolution

31

Confocal Microscopy

• Patented by Minsky in 1957

• Elimination the out of focus flare observed influorescence in thick sections

32

Confocal Microscopy Benefits

Suppression of out of focus light

The confocal detection pinhole acts as a barrier

To light outside the focal plane of the objective

Optical sectioning:

Specimen is monitored slice by slice (3D-resolution)

Each slice produces a sharp image by confocal optics

Improved resolution power:

Lateral resolution improved by appr. 1.4x.

Significant improvement in axial resolution

Improved contrast:

Rasterizing the specimen, straylight due to scattering is suppressed

33Point Spread Function (PSF)

Résolution

(Utilisation de billes pour obtenir

l’image d’une source ponctuelle

de lumière)

34

Diffraction

Single slit diffraction

http://phys.educ.ksu.edu/vqm/html/singleslit.html

35

XY Plane

Airy disc

Limited by Diffraction

D0 = 1.22 * l / NA (lateral)

XZ Plane

Résolution

36

Airy Disk formation

Airy Disk Formation

http://micro.magnet.fsu.edu/primer/java/imageformation/airydiskformation/index.html




37

Fluorescence Resolution

Res = 0.61*l / NA

credit: http://www.microscopyu.com/tutorials/

38

Imaging of small fluoresence beads

Vignette credit: http://www.microscopyu.com/tutorials/

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40



41

Resolution Widefiled vs Confocal

Credit : Prof. A. Diaspro

Conventional Confocal

42

Généralités

Introduction




Setup expérimental


Applications


Quantifications

Macros

Deconvolution (PSF)

Limitations


Calibration

Résolution

43

Confocal Implementation

Requires to excite and observe as small a

volume as possible on the sample.

This in turn requires a different way of

illumination and observation of the samples

Laser types

45

Confocal Beam Path

46

Confocal Principle

47

Scanning the Sample

48

Why Laser ?

• Intense light source confined to small beam size

Easily focused to diffraction limited spot

• Coherence is not required (can cause problems)

• As different fluorescence dyes have different

spectral characteristics, many laser lines are

required

49

Why scanning?

• Scanning in xy is necessary in order to form an image

• The scanning device is positioned between the beam

splitter and the objective

• The beam is scanned (made to move) on the excitation

side and descanned (made stationary) on the detection

side

50

Why a standard microscope ?

• Greatly facilitates the use of the system

• Provides a platform for mounting samples

• The application determines what type of microscope to be used

51

Microscope types

Inverted :

Live specimens

Upright : fixed samples

Fixed stage :

Patch clamping

52


53


54

AOTF (Acousto Optical Tunable Filter)

Module de sélection d’excitation

Source de lumière:

Laser multilinie ou

plusieurs lasers

couplés par fibre

• Libre sélection de ligne

• Contrôle indépendent de l‘intensité

sur chaque lineAOTF

55

Acousto Optical Beam Splitter:

Dichroïc Électronique

56

• Selectivité parfaite

•Transparence

le plus haut possible

• Plus “d’espace” pour

détecter les emissions

AOBS transmission

57


58

SP Detector - Advantages

• N'importe quelle spécification de filtre est

librement programmable

• Caractéristiques très étroite permet la

détection de plus de fluorescence

• Aucun élément filtre dans le trajet – plus de

transmission

• Balayage spectral avec haute résolution

(2-5nm, 200 steps)

59

Sectionnement optique de l‘échantillon

Suppression de la fluorescence en dehors du plan focal

Amélioration de la résolution latérale et axiale

Amélioration du contraste


The Digital Image

How is the optical image relayed on to a

screen

A digital image ?

61

Light Current

Photomultiplier Tubes (PMTs)

• High efficiency

• Wide spectral sensitivity

• Low dark current

• High dynamic range

62

Ca

tho

de

ra

dia

nt se

nsitiv

ity (

mA

/W)

--

--

Quantu

m e

ffic

iency(%

)

Longueur d’onde (nm)

100 200 600 700 800 900 1000300 400 500

0,1

1

10

100

Setup expérimental

Détection : photomultiplicateurs

Conversion d’une détection de

photons en signal électrique

63

APD

• higher sensitivity

• low background

• smaller dynamic range

• only a certain amount

of photons/time allowed

PMT

• lower sensitivity

• higher background

• bigger dynamic range

weak

samplesbright

samples

Setup expérimental

64

Current Digits (Digitazitation)

ADC Converter8bit / 12 bit (= 256 / 4096 Greyvalues)

Frame Store

Data Files (e.g. TIF)

About Data:

8 Sections 512x512 8bit 1ch: 2 MB

100 Sections 4096x4096 12 bit 5ch: 6000 MB

65

Digits Paper

Monitor

Printer

Slidemaker

PPT Präsentation

HTML Document

66

67

Objective Lens

Needs for confocal imaging:

• High aperture

• High colour correction

• Flat field

• Long working distance

• High transmittance

• Variety of coupling media

i.e. Plan Apochromat &

Plan-Fluotar

68

Objectif Glycérol :

Comparaison: Objectif à huile vs. Glycérol

yz

xz

Objectif Glycérol PL APO 63x1.3 Objectif huile PL APO 63x1.32

center section center section

Sample: muscle fibers embedded in Glycerol (80/20%) thickness ca. 100µm

69

Courbes de transmission des objectifs

70

Généralités

Introduction




Setup expérimental


Applications


Quantifications

Macros

Deconvolution (PSF)

Limitations


Calibration

Résolution

71

Paramétrage d’image

Mode de scan

Fréquence de balayage laser

Objectif utilisé

Zoom optique appliqué

Format d’image

Rotation de scan

Moyennage d’images

Accumulation

Gain du PMT

Offset du PMT

…

72

Immunohistochimie

© INRA Versailles IJPB LCC© INRA Versailles IJPB LCC

73

Généralités

Introduction




Setup expérimental


Applications


Quantifications

Macros

Deconvolution (PSF)

Limitations


Calibration

Résolution

74© INRA Versailles IJPB LCC

Immunohistochimie


Immunohistochimie


Immunohistochimie

77

l exc = 405 nm

l ém. = 422-496

l exc = 488 nm

l ém. = 496-533

l exc = 543 nm

l ém. = 556-628 nm

l exc = 633 nm

l ém. = 661-690 nm

T. Lecuit, Luminy, Marseille

Obj. 40X, 1.25

Zoom = 4,7

Embryon Drosophile

Immunohistochimie

78

Immunohistochimie

79

Immunohistochimie

80

Réflection

Etude de surface

81

Réflection

82

Time-Lapse

Arabidopsis thaliana

First channel: Cell wall in reflection.

2 & 3 channel: Monitoring mitochondrial (GFP-green) and plastid

(autofluorescence-red) movement.

22 fps

Courtesy of Prof. Dr. D. Menzel, Institut für Zelluläre und Molekulare Botanik

Zellbiologie der Pflanzen, Bonn University.

83

Fluorescence Recovery After Photobleaching

84

Région Bleachée

Région

adjacente

à la région

BleachéeI= f(t)

T. Lecuit, Luminy, Marseillelexc. = 488 nm

Obj. 63X, 1.32

Zoom = 4,6

Dt = 823 ms

Fluorescence Recovery After Photobleaching

85

Fluorescence Resonance Energy Transfer

86

Fluorescence Resonance Energy Transfer

Etude des modifications des

interactions moléculaires

Proximité donneur-accepteur 10–100 Å

Donor pre-bleach

Donor post-bleach

Acceptor pre-bleach

Acceptor post-bleach

Bleaching de l’accepteur

Augmentation de l’intensité de

fluorescence du donneur

87

Application FRET-AB

Setup pour imager

le donneur et l‘accepteur

Donneur

Accepteur

SP5 – LAS AF

88

Application FRET-AB

Setup du bleach

pour l‘accepteur

SP5 – LAS AF

89

Application FRET-AB

Quantification

Step 3

FRET-Efficiency

SP5 – LAS AF

90

Etudes Spectrales

Spectre

d’émission de

fluorescence

91

Etudes Spectrales

Spectres d’émission de fluorescence

92

Alexa 488 – GFP

Etudes Spectrales

Séparation spectrale

93

Example

FITC/TxR sample

2 channel recording:•Detection bands fine tuned

•No gaps between bands

•High efficient prism

•High efficient PMTs

•AOBS® applied

94

FITC

The total of all light collected from

FITC molecules will be distributed

into both channels.

We assume here:

¾ of all FITC emission go into the

green channel (G)

¼ of all FITC emission goes into the

red channel (R)

95

TxR

The total of all light collected from

TxR molecules will be distributed

into both channels.

We assume here:

1/5 of all TxR emission goes into the

green channel (G)

4/5 of all FITC emission goes into

the red channel (R)

96

Both dyes

In a real experiment, we will have

both dyes simultaneously in the

sample and therefore get signals

from both dyes in both channels.

97

A calculated measurement

TxR5

4FITC

4

1R

TxR5

1FITC

4

3G

98

Spectra: # and d

Important Note:

Theoretical spectrum: infinite number of datapoints.

Reality: maybe 50 channels, or just 2

Equidistant measurement is a good thing. But it is not always necessary.

In the unmix case, equidistance is no good: better to tune the bandwidth and

center wavelength. So we optimize S/N for separating data later.

Requires important hardware features as tunable bands, any filter

characteristic etc. which you find only with the Leica SP design.

99

What we dont‘t need:

Important note: it is sufficient to have two equations for

2 unknowns.

Those systems providing more and equidistant

channels (with gaps in between) are technically

massively inferior to the Leica SP AOBS concept.

TxR5

4FITC

4

1R

TxR5

1FITC

4

3G

This is just a set of n linear equations with n unkowns.

You may remember from primary school.

3y+2x=7

7y+ x=2

There are methods, to solve these by computers

100

Contributions

Unmixing is:

Solving sets of n linear

equations with n unknowns.

First proven records of

solutions go back some 4000

years (Egypt)

For a reference see:http://www.ETH\EducETH - Mathematik -

Leitprogramm Lineare

Gleichungssysteme.htm

101

Math Solution

1. Guess coefficients and subtract manually bleed through

Manual Dye Separation

2. Take n channel image, have pure dyes somewhere and let the

computer solve

Channel Dye Separation

3. Take a spectrum, have the spectra from the pure dyes and let the

computer solve

Spectral Dye Separation

102

Stat Solution

Let computer find best fitting line for clouds. Angle corresponds to

coefficients.

Adaptive Dye Separation

1. Let computer bend lines to coincide with axes

strong

2. Let computer bend until first non-empty bin hits the axis

weak

3. Find projection of n dimensions, when n dyes around

Dimensionality reduction (this is not an unmixing method)

103

Emission control:

SP performance test

Alexa 488 – GFP:

Almost complete

spectral overlap700650600

wavelength (nm)

550500450400

0

0,2

0,4

0,6

1

0,8

1,2

Alexa

GFP

104

1s 10s 12s 17s

32s 42s 52s 62s

GFP Photoactivable

Pulse laser IR 60 ms 800 nm

D. Choquet, Institut François Magendie, UMR 5091 CNRS, Bordeaux.Physiologie cellulaire de la synapse

105

Fluorescence Correlation Spectroscopy

Lien entre la diffusion de molécules et la fluctuation de l’intensité de

fluorescence dans un volume donné

Concentration

Coefficient de diffusion

Constante de dissociation

106

Fluorescence Correlation Spectroscopy

107

Généralités

Introduction




Setup expérimental


Applications


Quantifications

Macros

Deconvolution (PSF)

Limitations


Calibration

Résolution

108

Etude de colocalisation

Immunohistochimie

109

Fluorescence Lifetime IMaging FLIM

110

Fluorescence Lifetime IMaging FLIM

111

Probe: lexc/lem a (ns) b (ns)

BCECF 490/520 3.0 (acid) 3.8 (base)

Fluo-3

Lucifer Yellow

Sodium Green

Hoechst

490/520 2.44 (no Ca2+)

3.3

1.1 (low Na+)

2.2 (no acc., 7-AAD)

0.79 (Ca2+)

2.4 (high Na+)

1.4 (acceptor,7-AAD)

FITC

TRITC

490/520

543/

4.0 (pH > 7)

2.0

3.0 (pH < 3)

Rhodamine 700

Rhodamine 700

Cy3

Cy5

659/669

550/570

633/

1.6 (pH 9)

1.55 (H2O)

0.27

1.0

1.55 (pH 6)

2.99 (Ethanol)

0.5 (antibody conjug.)

GFP free (S65T)

CFP

YFP

488/507 2.68

1.3

3.7

Fluorophores and lifetimes

112

Fluorescence lifetime

fluo

rescenceex

cita

tio

n

S0

S1

add

ition

al pro

cesses

red

exponential decay e–t/

time after excitation

flu

ore

scen

ce s

ign

al

fluorescence lifetime:

• ave. time betw. excitation and emission

• characteristic property of dyes, ~ns

• depends on environment (ions, pH, …)

113

Fluorescence lifetime imaging (FLIM)

• measurement of lifetime with spatial resolution

• transformation of nanoseconds in color code

114

Leica TCS SP2 and D-FLIM

CFP-MHC

Cyan: CFP tagged to MHC(Major Histocompatibility Complex)

Before (A) and after (B) FLIM acquisition

1.25 3.5

Courtesy

Dan Davis, Imperial College London

A

B

Duration for acquisition: 200 s

405 nm 405 nm

115

Leica D-FLIM: CFP-MHC

A: Lifetime image

B: Lifetime distribu-

tion over the image

area

C: Fitted decay curve

(corresponding to the pixels selected by the blue cursor in A)

A

C

B

CFP tagged to MHC

116

Leica TCS SP2 and D-FLIM

FluoCells (BPAE)

Blue: 1.5 - 2.2 ns

Red: 2.2 - 2.6 ns

Green: 2.6 - 4.0 ns

Blue: DAPI (nucleus)

Red: Mitotracker Red (mitochondria)

Green: BODIPY FL phallacidin (actin)

405+488+543 nm 405 nm

Molecular Probes, F-14780

117

Leica D-FLIM: FluoCells

A B

C

A: Intensity image

B: Lifetime image

C: Fitted decay curve

(corresponding to the pixels selected by the blue cursor in A)

Blue: DAPI (nucleus)

Red: Mitotracker Red

(mitochondria)

Green: BODIPY FL

phallacidin (actin fibres)

118

Principe de l’excitation à deux photons

Processus de fluorescence en

excitation à deux photons

Deux photons d’énergie deux fois plus

faible (et donc de longueur d’onde deux

fois plus élevée) sont absorbés par la

molécule dans un laps de 10-16 s

hvex

2

S

1

S

0

hvem

Processus de fluorescence en

excitation à un photon

S

1

hvex hvem

S

0

E = hc/l

L’énergie d’un seul photon est absorbée

par un fluorochrome pour passer d’un

état d’énergie basal (S0) à un état excité

(S1)

Les caractéristiques du rayonnement émis par le fluorochrome en

excitation à deux photons sont inchangées

hvex

2

Perte d’énergie

vibrationnelle

Fluorescence

119

Plan

focal

En microscopie à balayage laser à deux photons, l’excitation est

strictement restreinte au volume focal

Fluorescence suite à une

absorption à un photon

Fluorescence suite à une

absorption à deux photons

120

Leica TCS STED

121

Multicolor Image of the NMJ

Liprin-a

STEDBruchpilot

confocal

1 µm500 nm

Courtesy of Prof. Stephan Sigrist

122

Resolution Increase by STED: Pure Physics!

confocalSTEDSTED +

deconvolution

STED is pure physics! But you can add mathematics on top!

123

Optical pathway

Confocal Profile

0 200 400 600 800 1000

x / nm

y

x

STED Profile

0 200 400 600 800 1000

x / nm

y

x

Typical lateral resolution: 200x200 nm Typical lateral FWHM in STED is 90x90 nm

Principle of STED microscopy

1. Excitation laser

2. Depletion laser

3. Original Excitation spot

4. Depletion (STED) ring

5. Effective excitation spot

6. Confocal pinhole

7. Detector

Formation of presynaptic

active zone (Liprin)

Courtesy S. Sigrist,

Wuerzburg

124

ACTIN

Confocal

STED

MICROTUBULES

Confocal

STED

125

The task

Microtubules of a

Vero cell

SOME EXAMPLES:

Neurophysiology (Synapse-cell-interactions, motoneurons etc.)

Endocytotic processes

Virus biology (Malaria, AIDS)

Pathology (Multiple Sclerosis etc.)

Increase xy resolution in fluorescence microscopy over

classical Abbe limits:

a

l

sin2

nd xy FWHMconfocal, xy: 200 nm

BUT: For numerous applications a higher resolution is required

-without the efforts and restrictions of an Electron microscope!-

126

The task

Microtubules of a

Vero cell

SOME EXAMPLES:

Neurophysiology (Synapse-cell-interactions, motoneurons etc.)

Endocytotic processes

Virus biology (Malaria, AIDS)

Pathology (Multiple Sclerosis etc.)

Increase xy resolution in fluorescence microscopy over

classical Abbe limits:

a

l

sin2

nd xy FWHMconfocal, xy: 200 nm

BUT: For numerous applications a higher resolution is required

-without the efforts and restrictions of an Electron microscope!-

127

Applications Example - Cell Biology

F-Actin -Tubulin

STED

Confocal

Nice for demonstrational purposes

128

Resolution enhancement by STED:

confocal

STED STED

STED STED STED

STEDSTEDSTED

STED

Excitation

spot

Depletion

ring

Overlaid

spots

Effective

spot

FWHM

ca. 250nmFWHM

ca. 90nm

A threefold improved resolution can make 9 spots out of 1 !

129

Superresolution Microscopy: Leica TCS 4Pi

130

The target of 4Pi microscopy

Increase axial resolution in fluorescence microscopy over

classical Abbé Limits

Inventor: Stefan Hell, Max Planck Institute for Nanobiophotonics,

Goettingen, Germany

a

l

sin

4.0

nFWHMxy

a

l

cos1

45.0

nFWHMz

xy resolution: ~ 200nm

z resolution (confocal): ~ 500nm

131

The solution: 4Pi microscopy

Confocal focus cross section 4Pi focus cross section

500 nm

4Pi microscopy combines the Numerical Aperture of 2 opposing

objectives through interference

The 4Pi focus volume is a factor 3 – 5 times smaller than the confocal focus

The axial resolution at the excitation wavelength of 780 nm is 110nm

Interference side lobes are removed by linear point deconvolution

x

z

132

Live yeast cell –

mitochondrial

protein distribution

Label: GFP

Objective: 63x1,2 W

Confocal

4Pi

4

m

Jörg Bewersdorf

MPI for Biophysical Chemistry

Göttingen

4Pi microscopy – spectacular 3D resolution

4

m

133

Central element: the 4Pi Interferometer

Illumination and imaging

from both sides, phase-

and wavefront-corrected

Two photon / confocal

system for scanning

134

Leica TCS 4Pi: complete system

135

Parasitology: Human red blood cell - healthy

Cell membrane

(Band-III protein)

labelled with

Qdot®-585 coupled

antibodies

4Pi – 3D rendering

Objective: Glycerol

100x/1,35 CORR

Cell diameter: ca. 8 m

Dr. J.A. Dvorak and Dr. F.Tokumasu,

National Institute of Allergy and Infectious Diseases, NIH, Washington (USA)

136

Parasitology: Human red blood cells -

healthy and Malaria infected - early stage

Band III – protein labelled

with QDot® 585-coupled

antibodies

Plasmodia (yellow, DNA)

stained with Hoechst 33258


Objective: Glycerol

100x/1,35 CORR

Cell diameters: ca. 8 m



137

Parasitology: Human red blood cell

Malaria infected – late stage

Band III – protein labelled

with QDot®-585

coupled antibodies

Plasmodia (yellow, DNA)

stained with Hoechst 33258


Objective: Glycerol

100x/1,35 CORR




138

Information exchange via the

Plasma Membrane is blocked

in mutated cell

Objective: 63x /1,20 W

Water immersion

Label: GFP


Immunology: MHC-II transport in B-Cells

Jörg Bewersdorf, MPI for Biophysical Chemistry, Göttingen,

You-Mi Kim, Havard Medical, Boston

Healthy cell

4Pi

Mutated cell

4Pi

139

Summary: Leica TCS 4Pi

Nov. 2003 Cover Page3D image of the mitochondrial compartment of a live yeast cell obtained through volume rendering of a superresolution 3D data stack recorded with a 4Pi

microscope. Courtesy of H. Gugel, R. Storz (Leica Microsystems CMS GmbH, Mannheim, Germany) and J. Bewersdorf, S. Jakobs, J. Engelhardt, S.W. Hell

(MPI for Biophysical Chemistry, Göttingen, Germany). The 3D image is superimposed on a colored high resolution scanning electron micrograph of

mitochondria and rough endoplasmic reticulum. © PhotoResearchers. Artwork rendered by Erin Boyle.

Commercial 4Pi microscope

delivers spectacular 3D resolution

Improved sub-cellular imaging

gives visual confirmation of

functional details

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SuperK - the White Light Laser

Martin Hoppe, Rolf BorlinghausLeica Microsystems CMS GmbH

Am Friedensplatz 3, 68165 MannheimGermany

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Today: merged set of conventional lasers excite specimen in a Confocal

Bulky construction of various combinations

Significant heat load

Inflexible

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Tomorrow: SuperK - the White Light Laser

Single solid state device

Adequate output power for imaging

Maintenance free

Low power consumption

Very flexible

Pulsed – FLIM source

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SuperK laser: Concept

Picosecond Laser

at 1060nm

Seed Source

Pump

Power Amplifier

Supercontinuum Fibre

Supercontinuum

Generation

White laser

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Supercontinuum Fibre

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Visible spectrum measured with AOTF

0

0,2

0,4

0,6

0,8

1

440 490 540 590 640 690 740

Wavelength (nm)

SuperK laser delivers full spectrum,

has tunable emission

Full tuneability

Optimal excitation

Minimal cross-excitation

450-740 nm range

AOTF

SuperK

Ilklk

n = 1..8

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SuperK laser: free tuning of up to 8 lines

simultaneously in intensity and wavelength

Freely tunable excitation

8-channel „dimmer“

THE light source

I

l

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High Quality Imaging with SuperK

Mouse Fibroblasts, triple staining

Cytoskeletal Protein Excitation Emission

Microtubuli 492 494-556

F-Actin 560 566-632

Vimentin 646 659-800

Documents

Start Pane Microscopie confocale - Télécom ParisTech€¦ · 4 Introduction Historique La microscopie photonique a été inventée au XVIIe siècle mais c'est à partir du XIXe