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Science Webinar Series
OPTICAL SECTIONING USING LIGHT SHEET MICROSCOPY
In Vivo Imaging with Astounding Resolution
15 June, 2012
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Science Webinar Series
OPTICAL SECTIONING USING LIGHT SHEET MICROSCOPY
In Vivo Imaging with Astounding Resolution
15 June, 2012
Brought to you by the Science/AAAS Custom Publishing Office
Participating Experts:
Ernst H. K. Stelzer, Ph.D.
Goethe University
Frankfurt, Germany
Pavel Tomancak, Ph.D.
Max Planck Institute of
Molecular Cell Biology and Genetics
Dresden Germany
Dresden,
Lars Hufnagel, Ph.D.
European Molecular Biology Laboratory
Heidelberg Germany
Heidelberg,
Sponsored by:
Reducing Photobleaching and Phototoxicity
in three
three--dimensional Imaging
Ernst H.K. Stelzer
Physical Biology
Buchmann Institute, FB 15 (IZN), CEF
CEF--MC
Goethe Universität Frankfurt am Main
June 2012
Physical Biology Group
Alexander Atzberger, Francesco Pampaloni,
N i
Nariman
A
Ansari,
i Roli
R li Richa
Ri h , Daniel
Richa,
D i l von
Wangenheim, Berit Langer, Kai Schwalm,
Schwalm, Christian
Mattheyer
Mattheyer,
y , Sabine Fischer, Sebastian Pfeiffer,
Pfeiffer, Bo
Bo--Jui
Chang, Frederic Strobel,
Strobel, Alexander Schmitz,
Michaela Koller, …
Some recent Alumni: F. Härle, P.
P. Theer
Theer,, H. Merkle,
Merkle, K. Khairy,
Khairy, S.
Rohr, P. Keller, U. Kržič,
Kržič, E. Reynaud, P. Girard, K. Greger,
Greger,
Swoger,
g , J. Huisken
Huisken,, A. Rohrbach, C. Engelbrecht,
Engelbrecht
g
,
H. Kress, J. Swoger
P. Seitz, T. Breuninger,
Breuninger, M. Neetz
Neetz,, L. Burger, A. Riedinger
Riedinger,, G. Ritter
UV Laser Cutter (UV Pulsed Laser Nano Scalpel) Julien Colombelli
(Barcelona) PFM (Optical Tweezers Based Photonic Force Microscope)
(Barcelona),
available as NanoTracker from JPK (Berlin, Germany).
Light SheetSheet-based Fluorescence
Mi
Microscopy (LSFM)
Physical Biology
Physics
Three-dimensional Imaging
ThreePh i l i l R
l
Physiological
Relevance
Instrumentation
Cell Biology
Developmental Biology
Motivation





Life sciences: alive
alive, threethree-dimensional,
dimensional multiple
processes as a function of time, relevant
Monitoring with a certain precision or
resolution over a certain period of time
Spatially & temporally resolved spectral
techniques are most appropriate
FP p
provide high
g intra vital specificity
p
y
Physiological context must be maintained
An azimuthal or  arrangement
An azimuthal or  arrangement
Advantages of a light sheet
In light sheet based fluorescence
microscopy
py ((LSFM)) the excitation and
the detection systems are separate and
operate along different directions
directions..
Advantages of a light sheet
In light sheet based fluorescence
microscopy
py ((LSFM)) the excitation and
the detection systems are separate and
operate along different directions
directions..
Advantages of a light sheet
In light sheet based fluorescence
microscopy
py ((LSFM)) the excitation and
the detection systems are separate and
operate along different directions
directions..
Advantages of a light sheet
In light sheet based fluorescence
microscopy
py ((LSFM)) the excitation and
the detection systems are separate and
operate along different directions
directions..
Advantages of a light sheet
In light sheet based fluorescence
microscopy
py ((LSFM)) the excitation and
the detection systems are separate and
operate along different directions
directions..
Confocal 
Microscopy
1992 - 2000
Advantages of a light sheet
In light sheet based fluorescence
microscopy
py ((LSFM)) the excitation and
the detection systems are separate and
operate along different directions
directions..
Confocal 
Microscopy
1992 - 2000
Advantages of a light sheet
In light sheet based fluorescence
microscopy
py ((LSFM)) the excitation and
the detection systems are separate and
operate along different directions
directions..
Advantages of a light sheet
In light sheet based fluorescence
microscopy
py ((LSFM)) the excitation and
the detection systems are separate and
operate along different directions
directions..
Advantages of a light sheet
In light sheet based fluorescence
microscopy
py ((LSFM)) the excitation and
the detection systems are separate and
operate along different directions
directions..
Advantages of a light sheet
Excitation and detection systems
are separate and run along
perpendicular directions.
directions.
Entire plane is excited and recorded
in p
parallel along
g an arbitrary
y
direction.
Planes in front or behind the light
sheet’s
sheet s plane experience no light
light.
Photo bleaching and photo toxicity
affect only the molecules in the
plane
l
th
thatt iis ill
illuminated
i t d and
d hence
h
observed..
observed
Only fluorophores in the light
sheet’s plane contribute to the
image, outout-ofof-focus blurr is heavily
reduced.
Advantages of a light sheet
Excitation and detection systems
are separate and run along
perpendicular directions.
directions.
Entire plane is excited and recorded
in p
parallel along
g an arbitrary
y
direction.
Planes in front or behind the light
sheet’s
sheet s plane experience no light
light.
Photo bleaching and photo toxicity
affect only the molecules in the
plane
l
th
thatt iis ill
illuminated
i t d and
d hence
h
observed..
observed
Only fluorophores in the light
sheet’s plane contribute to the
image, outout-ofof-focus blurr is heavily
reduced.
Chamber for LSFM
Light sheet illumination
optical
i l sectioning
i i
no damage outside light sheet
very low laser power
D t ti with
Detection
ith regular
l lens
l
focal plane overlaps light sheet
air / water immersion lens
variety of NA & magnification
Sample mounted e.g. in agarose
translation & rotation
in buffer
physiological conditions
Chamber
aqueous medium
minimized aberrations
environment
i
controllable
ll bl
Huisken, Swoger, Del Bene, Wittbrodt, Stelzer, Science 2004
Chamber for LSFM
Light sheet illumination
optical
i l sectioning
i i
no damage outside light sheet
very low laser power
D t ti with
Detection
ith regular
l lens
l
focal plane overlaps light sheet
air / water immersion lens
variety of NA & magnification
Sample mounted e.g. in agarose
translation & rotation
in buffer
physiological conditions
Chamber
aqueous medium
minimized aberrations
environment
i
controllable
ll bl
Huisken, Swoger, Del Bene, Wittbrodt, Stelzer, Science 2004
LSFM MultipleMultiple-View Imaging
Stacks of images
recorded by moving the
specimen
Common volume of
illumination light sheet
& detector
d t t ffocall plane
l
Rotation of specimen &
subsequent recording
of stack of images
LSFM MultipleMultiple-View Imaging
Stacks of images
recorded by moving the
specimen
Common volume of
illumination light sheet
& detector
d t t ffocall plane
l
Rotation of specimen &
subsequent recording
of stack of images
LSFM MultipleMultiple-View Imaging
Stacks of images
recorded by moving the
specimen
Common volume of
illumination light sheet
& detector
d t t ffocall plane
l
Rotation of specimen &
subsequent recording
of stack of images
LSFM MultipleMultiple-View Imaging
Stacks of images
recorded by moving the
specimen
Common volume of
illumination light sheet
& detector
d t t ffocall plane
l
Rotation of specimen &
subsequent recording
of stack of images
LSFM MultipleMultiple-View Imaging
Stacks of images
recorded by moving the
specimen
Common volume of
illumination light sheet
& detector
d t t ffocall plane
l
Rotation of specimen &
subsequent recording
of stack of images
Physical Biology
Physics
3D Imaging
Ph i l i l relevance
Physiological
l
Instrumentation
Cell Biology
Developmental Biology
Pampaloni, Reynaud, Stelzer (2007) Nature Reviews MCB,
MCB, 8:8398:839-845.
Verveer, ..., Stelzer (2007) Nature Methods,
Methods, 4:3114:311-313.
Pampaloni, Stelzer, Leicht, Marcello (2010) Proteomics.
Physical Biology
Physics
3D Imaging
Ph i l i l relevance
Physiological
l
Instrumentation
ThreeCell
Three-dimensional
Biology
Cell Biology
Developmental Biology
Pampaloni, Reynaud, Stelzer (2007) Nature Reviews MCB,
MCB, 8:8398:839-845.
Verveer, ..., Stelzer (2007) Nature Methods,
Methods, 4:3114:311-313.
Pampaloni, Stelzer, Leicht, Marcello (2010) Proteomics.
Cellular spheroids – LSFM imaging
X
Y
X
Z
Y
Z
Huisken, Swoger, Del Bene
Bene,, Wittbrodt, Stelzer (2004) Science 305:1007305:1007-1009.
Keller, Schmidt, Wittbrodt, Stelzer (2008) Science 322:1065-1069.
Keller, Schmidt, Santella, Khairy, Bao, Wittbrodt, Stelzer (2010) Nature Methods.
Physical Biology
Physics
Three-dimensional Imaging
ThreePh i l i l R
l
Physiological
Relevance
Instrumentation
Cell Biology
Developmental Biology
Plant Biology
Physical Biology
Physics
Three-dimensional Imaging
ThreePh i l i l R
l
Physiological
Relevance
Instrumentation
Cell Biology
Developmental Biology
Lateral root
growth
h
recording time 75 h,
15 min interval
detection: 20x/0.5
illumination: 5x/0.16
233 planes, 1.29 µm spacing
p35S::Lti6-GFP
488 nm & 525/45
0.55 mW, 100 ms
p35S::H2B-RFP
594nm & 628/40
1.5 mW, 100 ms
recording time 75 h
15 min interval
233 planes
1.29 µm spacing
detection:
20x/0.5 CZ W NAchroplan
illumination:
5x/0.16 CZ EC PlanNeofluar
p35S::Lti6-GFP
488 nm & 525/45
0.55 mW, 100 ms
p35S::H2B-RFP
594nm & 628/40
1.5 mW, 100 ms
Monolithic digital LSFM (mDSLM
mDSLM))
Physical Biology
Physics
Three-dimensional Imaging
ThreePh i l i l R
l
Physiological
Relevance
Instrumentation
Cell Biology
Developmental Biology
mDSLM Principal Components
mDSLM Principal Components
mDSLM Principal Components
dsmDSLM Optomechanics
dsmDSLM Optomechanics
dsmDSLM Optomechanics
dsmDSLM Optomechanics
LMG Instrumentation History
™
™
I was here
m
LMG Instrumentation History
™
™
now I am here
m
??
Co
ontact Detai
D
ils
Prof. Dr. Ernst H.K. Stelzer, Physical Biology
Buchmann Institute for Molecular Life Sciences (CEF
(CEF-MC)
MC)
Institute for Cell Biology and Neurosciences (IZN, FB 15)
Goethe University Frankfurt am Main (Campus Riedberg)
Max-von-Laue-Straße 15
D-60438
D
60 38 Frankfurt
rankfurt am Main, Germany
Phone: +49 (69) 798 42547, x42545 (Michaela Koller)
Fax: +49 (69) 798 42546
Mobile: +49 ((170)) 6357 168
ernst.stelzer@physikalischebiologie.de
stelzer@bio.uni-frankfurt.de
http://www.physikalischebiologie.de
http://www.researcherid.com/rid/A-7648-2011
http://www.eigenwelten.com/
p //
g
/
4th International LSFM Meeting 19-21 September 2012
Frankfurt am Main,
Main Germany
Organized by C. Lorenzo, E. Stelzer, P. Santi
Science Webinar Series
OPTICAL SECTIONING USING LIGHT SHEET MICROSCOPY
In Vivo Imaging with Astounding Resolution
15 June, 2012
Brought to you by the Science/AAAS Custom Publishing Office
Participating Experts:
Ernst H. K. Stelzer, Ph.D.
Goethe University
Frankfurt, Germany
Pavel Tomancak, Ph.D.
Max Planck Institute of
Molecular Cell Biology and Genetics
Dresden Germany
Dresden,
Lars Hufnagel, Ph.D.
European Molecular Biology Laboratory
Heidelberg Germany
Heidelberg,
Sponsored by:
Selective Plane Illumination
Mi
Microscopy
in
i developmental
d
l
t l biology
bi l
Pavel Tomancak
Max Planck Institute of Molecular Cell Biology and Genetics
D d
Dresden
Science webinar,
webinar June 2012
Genome
Information
Development
Cell
Behaviour
Divide
Move
Die
Genome
Information
Gene
Expression
p ess o
Development
Cell
Behaviour
Divide
Move
Die
From genome to pattern of gene expression
gene specific
probe
+
=
Images
Gene expression pattern database
RNA in situ projects
embryo
embryo
~8500 genes
G
Genome
Biology
Bi l
2007
~4000 genes
C ll 2007
Cell
Lecuyer & Krause
imaginal
discs
~6000 g
genes
unpublished
Tomancak & Dahmann
ovary
~3000 g
genes
unpublished
Helena Jambor
1D
3D
2D
4D
nD
The SPIM
(Selective Plane Illumination Microscope)
J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, Science 305, pp. 1007–1009, 2004.
SPIM examples
58
SPIM examples
5
9
Crustacean (Parhyale) multi-view SPIM reconstructions
Tassos
Pavlopoulos
DAY 3
DAY 4
DAY 5
DAY 8
Germ band
formation
Appendage
specification
Appendage
outgrowth
Differentiated
appendages
Registration problem
?
6
1
Preibisch et al. Nature Methods 2010
Bead based Registration
Stephan Preibisch
Stephan Saalfeld
SPIM registration as open source plugin
for Fiji
Preibisch et al. (2010) Nature Methods and et al. (2010) Nature Methods and Schindelin
Schindelin et al. Nature Methods in press
Isotropic SPIM imaging of Drosophila embryos
single view
multi view
xy
xz
yz
y
65
S1
Live imaging of development
5 minutes per 5-view frame
66
Drosophila Image Award 2011 finalist
OpenSPIM
price tag < 20,000 Euro
OpenSPIM build up
IKEA style assembly
Open Source Software to drive SPIM
SPIM in a suitcase
Photography by Vineeth Surendranath
Open hardware & software
Conclusions
Every developmental biologist should have a
SPIM
Conclusions
Every developmental biologist should have a
SPIM
High end set-ups for in toto imaging of
development
Conclusions
Every developmental biologist should have a
SPIM
High end set-ups for in toto imaging of
development
B d range, flflexible
Broad
ibl commercial
i l solution
l ti
Conclusions
Every developmental biologist should have a
SPIM
High end set-ups for in toto imaging of
development
B d range, flflexible
Broad
ibl commercial
i l solution
l ti
Custom, inexpensive and simple Open Source
set-ups
set
ups for parallelized imaging
Acknowledgements
Stephan Preibisch
Stephan Saalfeld
Peter Pitrone Johannes Schindelin Kevin Eliceiri
J H ik
Jan Huisken
my laboratory at the MPI‐CBG in Dresden
Science Webinar Series
OPTICAL SECTIONING USING LIGHT SHEET MICROSCOPY
In Vivo Imaging with Astounding Resolution
15 June, 2012
Brought to you by the Science/AAAS Custom Publishing Office
Participating Experts:
Ernst H. K. Stelzer, Ph.D.
Goethe University
Frankfurt, Germany
Pavel Tomancak, Ph.D.
Max Planck Institute of
Molecular Cell Biology and Genetics
Dresden Germany
Dresden,
Lars Hufnagel, Ph.D.
European Molecular Biology Laboratory
Heidelberg Germany
Heidelberg,
Sponsored by:
Light-sheet
Light
sheet microscopy:
From subcellular to organismal scales
Lars Hufnagel
EMBL Heidelberg
and BIOMS
Challenges in multi-scale imaging:
Drosophila embryonic development
~200μm
~500μm
Optical resolution: 0.3*0.3*1.0μ3m
8
Total number of 3D voxels: 2000x1000x200 = 4*10
Confocal imaging speed (1μsec/voxel): 400seconds
Problem: - too slow
- high
g p
phototoxicity/bleaching
y
g
- high background
MultiView-SPIM
MuVi-SPIM
MuVi
SPIM setup:
Main benefits:
• 2 illumination and 2 detection objectives
• 4 views without rotation
• Sample mounting from below
• 8 views with a single 90degree rotation
• Two high speed SCMOS cameras with 5.5MP • high speed (100fps with 5.5MP per image)
• enables realtime data registration and fusion
U. Krzic, T. Saunders, S. Guenther, S. Streichan, and L. Hufnagel, Nature Methods, 3 June 2012
MuVi-SPIM data registration and fusion
Image
registration
Image
fusion
Parameters given by the optical setup
and can be determined before the experiment
realtime data registration
and fusion
single high resolution
3D data set
Drosophila embryo development
I
Imaging
i setup:
t
• Stacks: 4x200 planes, spacing 1um
• Imaging time for 4 views: <10seconds
• Repeated
p
every
y 30seconds
• Total imaging time: 20hrs
• Total amount of data: 8TB
• 4 of 4 embryos hatched as larvae
MuVi-SPIM IT infrastructure
Realtime microscope control
High speed SCMOS
sensors
v
MuVi SPIM
MuVi-SPIM
1-2GByte/s
Data storage and processing
Camera computers &
data processing
10GBit/s
Optical fiber network
10GBit/s
Ultramicroscopy image from
Seidentopf and Zsigmondy, 1903
Tracking blastoderm nuclei
Nuclei tracked from division cycle 9-14
High speed of MuVi-SPIM allows for
faithful tracking of nuclei over extended periods
Stacks: 4x200 planes
planes, 1um spacing
of time and though divisions
divisions.
Imaging time for 4 views: < 5seconds
Repeated every 20seconds
Total imaging time: 3hrs
Drosophila membranes
Light-pad microscopy:
A quantitative look at protein dynamics
Light-pad dimensions
Optical properties
J. Capoulade, M. Wachsmuth, L. Hufnagel and M. Knop, Nature Biotechnology 2011
Quantitative imaging within cells and organs
Parallel recording of intensity fluctuations
Optical sectioning
5μm
Drosophila wing imaginal discs
Maps of protein concentrations and motility
Acknowledgments
Lab members
Furlong lab
• Tatjana Schneidt
• Christian Hoerner
• Justus Fueser
• Sebastian Streichan
• Uros Krzic
• Stefan Günther
• Timothy Saunders
• Gustavo de Medeiros
• Marvin Albert
• Balint Balazs
• Hilary Gustafson
SPIM FCS
SPIM-FCS
• Malte Wachsmuth
• Michael Knop
• Jérémie Capoulade
p
EMBL core facilities
• AMLF
• IT Services
• Mechanical workshop
Associated lab members
• Nils Norlin
• Céline Revenu
• Petr Strnad
More details
M
d il will
ill b
be posted
d on
www.spim.me
Nikon Microscopy, Andor Technology
Science Webinar Series
OPTICAL SECTIONING USING LIGHT SHEET MICROSCOPY
In Vivo Imaging with Astounding Resolution
15 June, 2012
Brought to you by the Science/AAAS Custom Publishing Office
Participating Experts:
Ernst H. K. Stelzer, Ph.D.
Goethe University
Frankfurt, Germany
Pavel Tomancak, Ph.D.
Max Planck Institute of
Molecular Cell Biology and Genetics
Dresden Germany
Dresden,
Lars Hufnagel, Ph.D.
European Molecular Biology Laboratory
Heidelberg,
Heidelberg Germany
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questions,
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and click
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Sponsored by:
Science Webinar Series
OPTICAL SECTIONING USING LIGHT SHEET MICROSCOPY
In Vivo Imaging with Astounding Resolution
15 June, 2012
Brought to you by the Science/AAAS Custom Publishing Office
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