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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
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
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.
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
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
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
Principe - Fluorescence
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
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
Principe de la microscopie confocale
Fluorescence – Sondes Fluorescentes et intérêts en biologie
Confocalité – avantages
Multiphoton
Setup expérimental
Paramétrage d‘images
Applications
3D / Reconstruction d‘images
Quantifications
Macros
Deconvolution (PSF)
Limitations
Etudes de colocalisation
Calibration
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)
36
Airy Disk formation
Airy Disk Formation
42
Généralités
Introduction
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
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
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
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
56
• Selectivité parfaite
•Transparence
le plus haut possible
• Plus “d’espace” pour
détecter les emissions
AOBS transmission
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
Microscopie confocale
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
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
70
Généralités
Introduction
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
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
…
73
Généralités
Introduction
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
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
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.
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
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
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.
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
107
Généralités
Introduction
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
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
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
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 !
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
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
4Pi – 3D rendering
Objective: Glycerol
100x/1,35 CORR
Cell diameters: ca. 8 m
Dr. J.A. Dvorak and Dr. F.Tokumasu,
National Institute of Allergy and Infectious Diseases, NIH, Washington (USA)
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
4Pi – 3D rendering
Objective: Glycerol
100x/1,35 CORR
Cell diameter: ca. 4 m
Dr. J.A. Dvorak and Dr. F.Tokumasu,
National Institute of Allergy and Infectious Diseases, NIH, Washington (USA)
138
Information exchange via the
Plasma Membrane is blocked
in mutated cell
Objective: 63x /1,20 W
Water immersion
Label: GFP
Cell diameter: ca. 8 m
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
140
SuperK - the White Light Laser
Martin Hoppe, Rolf BorlinghausLeica Microsystems CMS GmbH
Am Friedensplatz 3, 68165 MannheimGermany
141
Today: merged set of conventional lasers excite specimen in a Confocal
Bulky construction of various combinations
Significant heat load
Inflexible
142
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
143
SuperK laser: Concept
Picosecond Laser
at 1060nm
Seed Source
Pump
Power Amplifier
Supercontinuum Fibre
Supercontinuum
Generation
White laser
145
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
146
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