Opening doors to new worlds in Laser Scanning Microscopy
Welcome!
Workshop
University of Pennsylvania
March 21th 2002
Introduction to technique and
applications of
The
multi-fluorescence imaging
technology
for Laser Scanning Microscopy
Advanced Imaging Microscopy / Sebastian Tille
www.zeiss.com/lsm
1
Opening doors to new worlds in Laser Scanning Microscopy
Content
 Status Quo
 Open issues
 The new detection method
 Features and benefits
 Application examples
 A new way for FRET experiments
Advanced Imaging Microscopy / Sebastian Tille
www.zeiss.com/lsm
2
Opening doors to new worlds in Laser Scanning Microscopy
Focus on Life Sciencev
Developmental Biology
Neurobiology
Genetics
Molecules, organelles,
cells, tissue, embryos
as functional units
Physiology
Cell Biology
Goal:
- Non destructive observation of specimen
- Qualitative and quantitative analysis (2D, 3D, 4D, ...)
- Examine structure and function!
- Trends: fluorescence quantification .. FRAP .. FRET ..
Advanced Imaging Microscopy / Sebastian Tille
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3
Opening doors to new worlds in Laser Scanning Microscopy
Confocal Microscopy - Principle
Pinhole
Confocal
Sample
Advanced Imaging Microscopy / Sebastian Tille
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4
Opening doors to new worlds in Laser Scanning Microscopy
Brightfield versus Confocal
non confocal
Advanced Imaging Microscopy / Sebastian Tille
confocal
www.zeiss.com/lsm
5
Opening doors to new worlds in Laser Scanning Microscopy
We’ve come a long way…and keep on going!
Demands, demands, demands:
-
-
High resolution
- Spatial - x, y, z
- Temporal – Time lapse
- Spectral – Multi color
Authentic data
-
Convenience
-
Flexibility
It’s a
mammoth.
3D imaging, first successful try (whole mount):
Advanced Imaging Microscopy / Sebastian Tille
www.zeiss.com/lsm
6
Opening doors to new worlds in Laser Scanning Microscopy
Zeiss solutions before META
Proven functionality for (almost) any application:
LSM 510 <> LSM 5 PASCAL <> ConfoCor 2 <> LSM 510/ConfoCor 2 Combi
Flexible scanning strategies (DSP concept)
Multitracking
Fiber Input
Real ROIs
Multiple Pinholes
Spline Scan
Time Series
Physiology
Autofocus
Reuse-Concept
12 bit ADC
Image Database
UV
Image VisArt
....
Motorized Collimators
LSM 510 NLO (2photon)
Fluorescence Correlation Spectroscopy
Topography
...
Advanced Imaging Microscopy / Sebastian Tille
www.zeiss.com/lsm
7
Opening doors to new worlds in Laser Scanning Microscopy
The fluorescence emission
from the sample is separated
by dichroic beamsplitters
and/or by filters (longpass,
bandpass)
Long wavelength
Conventional Emission Separation
Short wavelength
Advanced Imaging Microscopy / Sebastian Tille
www.zeiss.com/lsm
8
Opening doors to new worlds in Laser Scanning Microscopy
Crosstalk distorts co-localization
FITC / Rhod
Simultaneous
Simultaneous
FITC
Rhod
merged
bovine endothelial cells
Advanced Imaging Microscopy / Sebastian Tille
www.zeiss.com/lsm
9
Opening doors to new worlds in Laser Scanning Microscopy
No Crosstalk with Multitracking
FITC / Rhod
Sequential
• Fast line-wise switching of laser lines and
•
Multitracking
•
•
FITC
Advanced Imaging Microscopy / Sebastian Tille
Rhod
intensities
Fast switching between tracks in line or
frame mode
Scanning quasi-simultaneously (in dual
directional scan)
Better signal with longpass filters (compared
merged
to bandpass)
www.zeiss.com/lsm
10
Opening doors to new worlds in Laser Scanning Microscopy
Multifluorescence Imaging
The Problem
 Spectral properties of the available dyes limit the experimental freedom.
 Often it is even difficult to clearly separate two fluorescence markers.
 With more markers, the problem grows increasingly complex.
(spectra published by Clonetech)
Cross-talk between the FP variants at the excitation and emisson level
Advanced Imaging Microscopy / Sebastian Tille
www.zeiss.com/lsm
11
Opening doors to new worlds in Laser Scanning Microscopy
Content
 Status Quo
 Open issues
 The new detection method
 Features and benefits
 Application examples
 A new way for FRET experiments
Advanced Imaging Microscopy / Sebastian Tille
www.zeiss.com/lsm
12
Opening doors to new worlds in Laser Scanning Microscopy
Acknowledgements
It has been a very fruitful collaboration with the people
at Caltech, who tried multi-spectral imaging
first with a prototyp using an LCTF setup.
Idea
- Profit from knowledge of remote sensing systems
- Instead of avoiding crosstalk – deal with it!
Jet Propulsion
Laboratory
Rusty Lansford
Scott Fraser
Mary Dickinson
Advanced Imaging Microscopy / Sebastian Tille
Greg Bearman
www.zeiss.com/lsm
13
Opening doors to new worlds in Laser Scanning Microscopy
The new detection scheme




Efficient PMT array with 32 elements
Special grating as dispersive medium
Covering entire emission spectral range
Adjustable pinhole (x,y, diameter)


Replaces one conventional channel
Upgradeable


Fast multiplexed electronic selection
of PMT elements/combinations
Patents pending
Advanced Imaging Microscopy / Sebastian Tille
www.zeiss.com/lsm
14
Opening doors to new worlds in Laser Scanning Microscopy
Look inside the solution – LSM 510 META






Based on the proven LSM 510 concept
Multiple pinhole concept
Adjustable pinholes (x,y, Ø)
Efficient beam path
Plus: META detector
 PMT array with 32 elements
 Reflection grating for even dispersion
 Capture full emission spectra
Advanced Imaging Microscopy / Sebastian Tille
www.zeiss.com/lsm
15
Opening doors to new worlds in Laser Scanning Microscopy
System setup for life science
LSM 510 META
Advanced Imaging Microscopy / Sebastian Tille
www.zeiss.com/lsm
16
Opening doors to new worlds in Laser Scanning Microscopy
Advantages of the new technique (1)

Electronic band selection => no moving parts
Highly reproducible
Settings can be stored and reused
Highly stable
Grating is temperature insensitive
Real - Rapid - Reproducible - Reliable
Advanced Imaging Microscopy / Sebastian Tille
www.zeiss.com/lsm
17
Opening doors to new worlds in Laser Scanning Microscopy
Advantages of the new technique (2)

Fast linewise Multitracking with user defined emission bands
 More signal by using long pass settings
 Crosstalk free data (prerequisite for colocalization)
 Faster than framewise switching
(better suited for live cell imaging)
 More than 4 dyes possible
METATRACKING . . . .
Advanced Imaging Microscopy / Sebastian Tille
www.zeiss.com/lsm
18
Opening doors to new worlds in Laser Scanning Microscopy
Advantages of the new technique (3)

Fast electronic lambda acquisition
Parallel acquisition of spectral data for all pixel
Collects Lambda Stack (xy-l) with user definable
number of elements
Get the knowledge of the spectral conditions
of individual pixels or defined areas (ROIs)
Optimize channel setup for scanning accordingly
Advanced Imaging Microscopy / Sebastian Tille
www.zeiss.com/lsm
19
Opening doors to new worlds in Laser Scanning Microscopy
Emission Fingerprinting - 3 easy steps
1.
Acquire a single Lambda Stack or a series of Lambda stack(s)
Advanced Imaging Microscopy / Sebastian Tille
www.zeiss.com/lsm
20
Opening doors to new worlds in Laser Scanning Microscopy
Emission Fingerprinting - 3 easy steps
1.
2.
Acquire a single Lambda Stack or a series of Lambda stack(s)
Select Regions of Interest (ROI) in the Lambda stack(s) or
load reference spectra from Spectra Database
Advanced Imaging Microscopy / Sebastian Tille
www.zeiss.com/lsm
21
Opening doors to new worlds in Laser Scanning Microscopy
Emission Fingerprinting - 3 easy steps
1.
2.
3.
Acquire a single Lambda Stack or a series of Lambda stack(s)
Select Regions of Interest (ROI) in the Lambda stack(s) or
load reference spectra from Spectra Database
Unmix
Advanced Imaging Microscopy / Sebastian Tille
www.zeiss.com/lsm
22
Opening doors to new worlds in Laser Scanning Microscopy
Discover spectral signatures within ROIs
ln
l3
l1
l2
Advanced Imaging Microscopy / Sebastian Tille
I
l1 l2 l3 l4
www.zeiss.com/lsm
.....
ln
23
Opening doors to new worlds in Laser Scanning Microscopy
Which cells express CFP, GFP and YFP?
Cell 1
Cell 2
Cell 3
1400
1200
1000
800
600
400
200
0
420 430 440 450 460 470 480 490 500 510 520 530 540 550 560 570 580
GFP
CFP
YFP
Cell 1 = CFP
Cell 2 = GFP
Cell 3 = YFP
M. Dickinson, R. Lansford, S. Fraser,
BIC, Caltech
Advanced Imaging Microscopy / Sebastian Tille
www.zeiss.com/lsm
24
Opening doors to new worlds in Laser Scanning Microscopy
Linear Unmixing – brief explanation
Linear unmixing can determine the relative weights (abundance) of component spectra even when the individual spectra overlap
+
YFP
450
500
550
450
600
+
CFP
500
550
600
GFP
450
500
550
600
.4*CFP+.4*GFP+.2*YFP
.5*GFP+.5*YFP
G. Bearman, JPL
450
Advanced Imaging Microscopy / Sebastian Tille
500
550
600
www.zeiss.com/lsm
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Opening doors to new worlds in Laser Scanning Microscopy
META + Emission Fingerprinting succeed!
Insufficient separation
using (variable) band pass detection
Advanced Imaging Microscopy / Sebastian Tille
Crosstalk-free separation
using Emission Fingerprinting
www.zeiss.com/lsm
26
Opening doors to new worlds in Laser Scanning Microscopy
Principle of Linear Unmixing
=a×
+b×
GFP
Advanced Imaging Microscopy / Sebastian Tille
YFP
www.zeiss.com/lsm
27
Opening doors to new worlds in Laser Scanning Microscopy
Content
 Status Quo
 Open issues
 The new detection method
 Features and benefits
 Application examples
 A new way for FRET experiments
Advanced Imaging Microscopy / Sebastian Tille
www.zeiss.com/lsm
28
Opening doors to new worlds in Laser Scanning Microscopy
Multi-fluorescence with Metatracking
Example - Metatracking:
DAPI, Alexa Fluor 488, Cy3, Cy5
Developing eye of the zebrafish;
Green - cell adhesion molecule Tag-1 (Alexa Fluor 488)
Red - tubulin (Cy3)
Purple - sugar epitope PSA (Cy5)
Blue - cell nuclei (DAPI)
Track 1 (488/633)
Track 2 (364 /543)
METATRACKING the enhanced Multitracking
Dr. M. Marx, Prof. M. Bastmeyer, University of Konstanz, Germany
Advanced Imaging Microscopy / Sebastian Tille
www.zeiss.com/lsm
29
Opening doors to new worlds in Laser Scanning Microscopy
The issue: FP’s spectra do overlap!
Fluorescent Proteins are essential for life science studies.
However, overlapping emission AND excitation spectra and
corresponding crosstalk makes combinations difficult for imaging!
(especially true for multiphoton imaging)
Data from Clonetech, Inc.
www.clonetech.com
Advanced Imaging Microscopy / Sebastian Tille
Heavy overlap!
www.zeiss.com/lsm
30
Opening doors to new worlds in Laser Scanning Microscopy
Separating 2 FPs – GFP and YFP
Example 2 – Emission Fingerprinting:
GFP and YFP (Distance of emission peaks approx. 12nm)
Human epidermoid tumor cells A431 expressing GFP and a YFP-Rab11 fusion protein
Sample: Jochen Rink, Max Planck institute for Cell Biology and Genetics, Dresden, Germany
Advanced Imaging Microscopy / Sebastian Tille
www.zeiss.com/lsm
31
Opening doors to new worlds in Laser Scanning Microscopy
Separating 2 FPs – GFP and YFP
Example 2 – Emission Fingerprinting:
GFP and YFP (Distance of emission peaks ca. 12nm)
A431 cells expressing GFP, Rab11-YFP
GFP
Advanced Imaging Microscopy / Sebastian Tille
YFP
www.zeiss.com/lsm
overlay
32
Opening doors to new worlds in Laser Scanning Microscopy
Separating 3 FPs – CFP, GFP, YFP
Examples 3 and 4 – Emission Fingerprinting:
CFP, GFP and YFP
• Cultured cells expressing CFP-RanGAP1, GFP-emerin und YFP-SUMO1
• Mixture of NIH3T3 cells expressing either CFP, GFP or YFP
Advanced Imaging Microscopy / Sebastian Tille
www.zeiss.com/lsm
33
Opening doors to new worlds in Laser Scanning Microscopy
Separating 3 FPs – CFP, GFP, YFP
Examples 3 and 4 - Emission Fingerprinting:
CFP, GFP and YFP
• Cultured cells expressing ECFP-RanGAP1, EGFP-emerin und EYFP-SUMO1
• Mixture of NIH3T3 cells expressing either CFP, GFP or YFP
CFP
GFP
YFP
CFP, GFP,
YFP
Courtesy: Prof. Y. Hiraoka, KARC, Kobe, Japan;
Mary Dickinson, PhD, Caltech, Pasadena, USA
Advanced Imaging Microscopy / Sebastian Tille
www.zeiss.com/lsm
34
Opening doors to new worlds in Laser Scanning Microscopy
High Overlap - Separating GFP and FITC
Example 5 – Emission Fingerprinting:
GFP and FITC (Distance of emission peaks 7nm)
Cultured fibroblasts expressing a GFP-Histone2B fusion protein,
actin filaments stained with FITC-phalloidin
Excitation: 488nm
Emission
Fingerprinting
Emission: bandpass
505-530nm
FITC
GFP
Advanced Imaging Microscopy / Sebastian Tille
www.zeiss.com/lsm
35
Opening doors to new worlds in Laser Scanning Microscopy
Emission Fingerprinting – GFP & FITC
Example 5 – Emission Fingerprinting:
GFP and FITC (Distance of emission peaks 7nm)
Cultured fibroblasts expressing a GFP-Histone2B fusion protein (green),
actin filaments stained with FITC-phalloidin (red)
GFP
FITC
Clear separation of signals despite
heavy spectral and spatial overlap
of emission spectra
Sample: Mary Dickinson, PhD, Caltech, Pasadena, USA
Advanced Imaging Microscopy / Sebastian Tille
www.zeiss.com/lsm
36
Opening doors to new worlds in Laser Scanning Microscopy
Ultrahigh Overlap – Sytox Green & FITC
Example 6 – Emission Fingerprinting:
Sytox Green (nuclei) and FITC (actin filaments) in cultured fibroblasts
Sample: Mary Dickinson, PhD, Caltech, Pasadena, USA
Advanced Imaging Microscopy / Sebastian Tille
www.zeiss.com/lsm
37
Opening doors to new worlds in Laser Scanning Microscopy
Emission Fingerprinting – Example
Example 6 – Emission Fingerprinting:
Sytox Green (nuclei) and FITC (actin filaments) in cultured fibroblasts
FITC
Sytox Green
overlay
Sample: Mary Dickinson, PhD, Caltech, Pasadena, USA
Advanced Imaging Microscopy / Sebastian Tille
www.zeiss.com/lsm
38
Opening doors to new worlds in Laser Scanning Microscopy
Separating 4 FPs – CFP, CGFP, GFP, YFP
Example 7:
CFP, CGFP, GFP and YFP
Cultured cells expressing 4 FPs in ER, nuclei, plasma membranes and mitochondria, repectively
Sample: Drs. Miyawaki, Hirano, RIKEN, Wako, Japan
Advanced Imaging Microscopy / Sebastian Tille
www.zeiss.com/lsm
39
Opening doors to new worlds in Laser Scanning Microscopy
Emission Fingerprinting – 4x FP challenge
Example 7– Emission Fingerprinting:
CFP, CGFP, GFP and YFP
Cultured cells expressing 4 FPs in ER, nuclei, plasma membranes and mitochondria, respectively
(can be excited with single wavelength from either single or multiphoton laser)
CFP
CGFP
GFP
YFP
Sample: Drs. Miyawaki, Hirano, RIKEN, Wako, Japan
Advanced Imaging Microscopy / Sebastian Tille
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40
Opening doors to new worlds in Laser Scanning Microscopy
FRAP & Emission Fingerprinting
Example 8:
FRAP with Emission Fingerprinting
ROI 1
Experiment: Pixel-precise bleaching of GFP and YFP
GFP
ROI 2
ROI 2
ROI 1
Result:
Slow flow-back of GFP from the cytoplasm and adjacent nucleus
into the bleached nucleus; free GFP passes the nuclear membrane
YFP
Sample: Dr. F. Boehmer, Friedrich Schiller University Jena, Germany
Advanced Imaging Microscopy / Sebastian Tille
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41
Opening doors to new worlds in Laser Scanning Microscopy
Eliminate background
Tissue section of the adrenal gland
stained with Alexa 488
conjugated antibody
3000
2500
2000
1500
1000
500
0
509
519
529
539
section background
Advanced Imaging Microscopy / Sebastian Tille
www.zeiss.com/lsm
549
559
569
Alexa 488
42
Opening doors to new worlds in Laser Scanning Microscopy
FRET - Basics
Yellow Cameleon 2 as internal Ca2+-sensor
Can be expressed in living cells as internal Ca2+-reporter
low Ca2+ no FRET
high Ca2+
FRET (conformation change)
A. Miyawaki, RIKEN, Wako, Japan
Advanced Imaging Microscopy / Sebastian Tille
www.zeiss.com/lsm
43
Opening doors to new worlds in Laser Scanning Microscopy
FRET with LSM 510 META
Advantage for META detection over standard bandpass imaging
Conventional FRET acquisition
Lambda Stack Acquisition
Emission
Emission
no FRET
FRET
460
480
500
520
540
560
580
600
460
480
Wavelength (nm)
• Detection of two narrow emission bands
• Some signal is discarded
Advanced Imaging Microscopy / Sebastian Tille
500
520
540
560
580
600
Wavelength (nm)
• Complete emission detection
• No signal is discarded
www.zeiss.com/lsm
44
Opening doors to new worlds in Laser Scanning Microscopy
FRET & Emission Fingerprinting
Step 1: Aquire complete emission via Lambda Stack acquisition
Series of Lambda Stacks acquired over time (xylt)
0s
100 s
200 s
300 s
Y. Hiraoka, KARC, Kobe, Japan/ A. Miyawaki, RIKEN, Wako, Japan
Cytoplasmic expression of Yellow Cameleon2 in a cultured cell
Advanced Imaging Microscopy / Sebastian Tille
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45
Opening doors to new worlds in Laser Scanning Microscopy
FRET & Emission Fingerprinting
Step 2: Linear Unmixing via Spectral Signatures and Reference Spectra
Reference spectra
Intensity
Stimulus
YFP
FRET
CFP
Time (s)
Y. Hiraoka, KARC, Kobe, Japan/ A. Miyawaki, RIKEN, Wako, Japan
Cytoplasmic expression of Yellow Cameleon2 in a cultured cell
Advanced Imaging Microscopy / Sebastian Tille
www.zeiss.com/lsm
46
Opening doors to new worlds in Laser Scanning Microscopy
FRET & Emission Fingerprinting
Step 3: YFP/CFP ratio
Reference spectra
YFP/CFP ratio
Intensity
Stimulus
FRET
Time
Higher information density
...Lambda Stacks

Y. Hiraoka, KARC, Kobe, Japan/ A. Miyawaki, RIKEN, Wako, Japan
Advanced Imaging Microscopy / Sebastian Tille
www.zeiss.com/lsm
47
Opening doors to new worlds in Laser Scanning Microscopy
More Advantages with META

Combines scanning flexibility with enhanced detection possibilities

Combination of Conventional & Multispectral Imaging
(META is an additional detector – nothing is taken away!)

Superior technology driven by application needs





Multiple pinholes benefits
Fast excitation & emission Multitracking - Metatracking
Fast electronic Lambda Stack acquisition
Adding the spectral dimension, xyz-t-l
Clear spearation of channels with Emission Fingerprinting

Upgradeable

Increased experimental freedom for upcoming applications




Multi FP imaging
Separation of background or autofluorescence
Perfectly suited for single and multiphoton excitation
Multi-FRET!
Advanced Imaging Microscopy / Sebastian Tille
www.zeiss.com/lsm
48
Opening doors to new worlds in Laser Scanning Microscopy
Opening doors to new worlds!
Thank you for your attention!
Advanced Imaging Microscopy / Sebastian Tille
www.zeiss.com/lsm
49
Opening doors to new worlds in Laser Scanning Microscopy
More Information!




Laser Scanning Microscopy
 www.zeiss.de/lsm
Fluorescence Correlation Spectroscopy
 www.zeiss.de/fcs
Mirrored at
 www.zeiss.com
Workshop on FCS
 sponsored by Zeiss in May 21st, 22nd, St. Louis, USA
 register via internet!
Advanced Imaging Microscopy / Sebastian Tille
www.zeiss.com/lsm
50
Opening doors to new worlds in Laser Scanning Microscopy
Sensitivity comparison (green emission)
META
PMT 2
Sytox Green and
Fitc-Phalloidin
Gain=900
458 EX
502-545nm
Advanced Imaging Microscopy / Sebastian Tille
Sytox Green and
Fitc-Phalloidin
Gain=1250
458 EX
505-550nm BP
www.zeiss.com/lsm
51
Opening doors to new worlds in Laser Scanning Microscopy
Sensitivity comparison (red emission)
META
PMT 3
ToPro-3
Gain=900
543 EX
587-800nm
Advanced Imaging Microscopy / Sebastian Tille
ToPro-3
Gain=1250
543 EX
585nm LP
www.zeiss.com/lsm
52
Opening doors to new worlds in Laser Scanning Microscopy
How much overlapping is resolvable?
Fluorochrome
Emission Peak
DiOa
506nma
eGFPb
507nmb
Alexa 488a
519nma
Fluoresceina
519nma
Oregon Greena
526nma
Sytox Greena
524nma
ToPro-1a
531nma
a Molecular Probes, Eugene, OR b Clonetech
Advanced Imaging Microscopy / Sebastian Tille
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53
Opening doors to new worlds in Laser Scanning Microscopy
Green dyes with close emission peaks
120
120
100
100
80
80
60
60
40
40
20
20
0
490
500
510
520
530
540
GFP
550
560
570
580
507
0
490
510
520
DiO
530
Alexa488
120
120
100
100
80
80
60
60
40
40
20
20
0
490
500
540
550
560
570
519580
Fitc-Phall
0
500
510
520
530
OreGrn
540
SytoxGrn
Advanced Imaging Microscopy / Sebastian Tille
550
560
570
580
525
490
500
510
520
530
540
ToPro1
www.zeiss.com/lsm
550
560
570
580
531
54
Opening doors to new worlds in Laser Scanning Microscopy
Shades of Green
120
100
80
60
40
20
0
490
500
510
OreGrn
Advanced Imaging Microscopy / Sebastian Tille
520
GFP
530
DiO
540
Fitc-Phall
550
Alexa488
www.zeiss.com/lsm
560
SytoxGrn
570
580
ToPro1
55
Opening doors to new worlds in Laser Scanning Microscopy
Results of pairwise comparison
Alexa488
DiO
FITC
GFP
Ore Green
Sytox
ToPro1
Alexa488
DiO
Not
Attempted
FITC
NO
Not
Attempted
GFP
YES
NO
YES
Ore Green
YES*
Not
Attempted
YES*
YES
Sytox
YES*
YES
YES*
YES
NO
ToPro1
YES
YES
YES
Not
Attempted
YES
YES
* Probe concentration must be balanced
Advanced Imaging Microscopy / Sebastian Tille
www.zeiss.com/lsm
56
Opening doors to new worlds in Laser Scanning Microscopy
Colors…
Whatever comes next…
new XFP variants?
CGFP
Alexa
350
CFP
Alexa
488
Alexa
543
Alexa
568
GFP
YFP
DsRed
DiA
DAPI
Hoechst
Advanced Imaging Microscopy / Sebastian Tille
DiI
Fluorescein
Oregon green
DiO
www.zeiss.com/lsm
Rhodamine
Texas Red
DiD
57
Opening doors to new worlds in Laser Scanning Microscopy
Two-photon excitation
Whatever comes next…
Alexa
350
Fluorochromes
Excited
at 830nm
Alexa
488
CGFP
CFP
Alexa
568
new XFPs variants?
DsRed
YFP
GFP
DiA
DAPI
Hoechst
Advanced Imaging Microscopy / Sebastian Tille
Alexa
543
Fluorescein
Oregon green
DiO
www.zeiss.com/lsm
DiI
Rhodamine
Texas Red
DiD
58
Opening doors to new worlds in Laser Scanning Microscopy
Multi-dimensional analysis of living cells
X, Y and t
X, Y and Z
tn
zn
z3
t2
z2
z1
t3
t1
X, Y and l
ln
l3
3 spatial dimensions
1 time dimension
multiple colors, lifetimes,
intensity
+ molecular genetic analysis
l2
l1
Advanced Imaging Microscopy / Sebastian Tille
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59