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Sponsored by:
Innovations in Light Sheet
Microscopy
Strategies and New Applications
October 29, 2014
Brought to you by the Science/AAAS Custom Publishing Office
Participating Experts
Thai Truong, Ph.D.
University of Southern California
Los Angeles, California
Pete Pitrone, DipRMS
MPI-CBG
Dresden, Germany
Webinar Series
Innovations in Light Sheet
Microscopy
Strategies and New Applications
October 29, 2014
Sponsored by:
Orla Hanrahan, Ph.D.
Andor Technology
Belfast, Ireland
Innovations in
Light Sheet Microscopy: Strategies and New Applications
Thai Truong, Ph.D. University of Southern California, Los Angeles, CA
Peter Gabriel Pitrone DipRMS Max Planck Institute of Molecular Cell Biology and Genetics
Dr. Pavel Tomancak research group
2014/10/29 – Science Magazine Webinar
R. Hooke
Brief history of optical microscopy
• 2nd century BC: Measurement of light refraction
• 1st century AD: First glass lenses, magnifier
• Early 17th cent.: First microscope, Janssen
• 1665: Publication of Micrographia, Hooke
Observation of the first “cell” in cork
Arguably the birth of modern biology
• 1911-1913: First fluorescence microscope, Heimstaedt & Lehmann
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Parallelization allows imaging multiple points simutaneously
STED, PALM, STORM sub-diffraction resolution
Single-point scanning improves sectioning
trade-off
Point laser scanning microscopy (1 or 2 photon)
Spinning-disk confocal
Competing performance parameters of biological optical imaging
Resolution Depth
Speed
Manipulate ON/OFF of single fluorophores
Parallelization allows imaging multiple points simutaneously
Single-point scanning improves sectioning
trade-off
Point laser scanning microscopy (1 or 2 photon)
Spinning-disk confocal
Resolution
Manipulate ON/OFF of single fluorophores
Depth
Speed
Photodamage
• Bleaching
• Toxicity
Signal to noise ratio
photons ofNumber
STED, PALM, STORM sub-diffraction resolution
Competing performance parameters of biological optical imaging
Light Sheet Microscopy
Graphic from Huisken & Stanier, Development (2009)
OpenSPIM: An easy-to-build modular open-source light sheet
fluorescence microscope for life scientists
2014/10/29 - Science Magazine Webinar
Peter Gabriel Pitrone DipRMS Max Planck Institute of Molecular Cell Biology and Genetics
Dr. Pavel Tomancak research group
Ultramicroscope - 1903
Henry Seidentopf built the slit ultramicroscope with the help of Richard Zsigmundy at the turn of the 20th century. Zsigmundy later won the Nobel prize in Chemistry in 1925 for his work on colloids.
Image credit: corporate.zeiss.com “Technical Milestones of Microscopy”
Fluorescence microscope - 1908
Seidentopf then went on to work with August Köhler on a novel transmitted light fluorescence microscope only 5 years later. Why did they not incorporate both systems together to make a light sheet fluorescence microscope?! Lack of the correct technology.
Image credit: corporate.zeiss.com “Technical Milestones of Microscopy”
“No power on earth can stop an idea whose time has come” – Victor Hugo
Three pieces of hardware that were needed but
not yet available at the time:
1.Coherent light source such as a laser - 1960
2.Digital array detectors such as CCDs – 1969
3.Computers to store the data - 1970 (or later)
“No power on earth can stop an idea whose time has come” – Victor Hugo
Other pieces of the puzzle missing at the
time:
• Fluorescent markers - dyes & proteins
• Expressing these markers in vivo
The idea’s time had come
Ernst Stelzer put all the pieces together with the help of Jan Huisken in 2004 - a full century later. This year marks the 10 year anniversary of this discovery.
Image Credit: Stelzer lab website
Selective Plane Illumination Microscopy (SPIM)
Benefits of 3D sample suspension and horizontal oriented optics
Benefits of camera detection
• Imaging speed – limited only by camera rate
• Quantum efficiency – sensitivity to dim signals
• Full field of view – in one shot
Image Credit: Ulrik Günther
Two orthogonal views showing cardiac contractions in a 5 day old zebrafish heart expressing Tg(myl7:DsRed, kdrl:GFP). (Michaela Mickoleit et al, 2014) Processed.
Benefits of light sheet illumination little to no photo damage
Zeiss Lightsheet Z.1 dataset of a developing Drosophila melanogaster embryo expressing His-YFP in all the cell nuclei, 715 time points, 2 sided illumination, 5 views, 1 minute 20 seconds for 18 hours, 4TB
OpenSPIM – DIY microscope
Image Credit: Michael Weber
OpenSPIM imaging large samples
2 day old Zebrafish expressing Tg(Bactin:H2A-EGFP) in all cell nuclei, 6 tiled Z stacks of 200 planes each, stitched using Grid/Collection Fiji plug-in.
OpenSPIM imaging fast processes
2 day old Zebrafish expressing Tg(cmlc2:EGFP) in the walls of the heart. Left: overlay of transmitted light and fluorescence signal. Right: only fluorescence.
OpenSPIM multi-view imaging
Drosophila melanogaster expressing Csp-sGFP in the central nervous system, 5 views 72°, 150 planes per view, 6 minute time points, 14 hours 40 minutes.
OpenSPIM wiki
http://www.openspim.org
Parts lists
OpenSPIM assembly videos
OpenSPIM assembly animations
OpenSPIM can be built in as little as 19 steps, once all the smaller assemblies are put together.
µManager Acquisition Software
OpenSPIM µManager Plug-in The OpenSPIM plug-in gives the user the capability to: • Position the sample in the
X, Y, Z, & rotation (Y) axes
• List position ranges for multi-view time-lapses
• Image one single position as fast as the camera and data connection allow
Benefits of µManager: Support of Various Hardware
Side by side camera test done by Daniel Holland at the EMBO practical course on Light sheet microscopy in Dresden Germany 2014
SPIM in a Carry-on Container
Image Credit: Vineeth Surendranath
This OpenSPIM system has been on two flights in the past year and a half, with out “much” trouble…
OpenSPIM contributors
Image Credit: Vineeth Surendranath
Johannes Schindelin Luke Stuyvenberg Kevin Eliceiri Jan Huisken Stephan Preibisch Michael Weber Ulrik Günther
Pavel Tomancak and Peter Gabriel Pitrone
Survey of latest developments in light sheet microscopy
• Excitation
• Detection
• Applications
Graphic from Huisken & Stanier, Development (2009)
Excitation: how to create the illumination light sheet?
• Cylindrical optics:
static, real sheet
• Circular-focused Gaussian beam, scanned
virtual sheet
better light throughput
better spatial/temporal control of light
Keller et al., Science (2008)
Graphic adapted from Gao et al., Nat. Protoc. (2014)
High NA
Gaussian beam
Low NA
Gaussian beam
High NA
Bessel beam
Excitation: thinner sheet, larger field of view, less photodamage
Trade-off: thinner sheet requires higher NA, leads to smaller field of view
Graphic adapted from Gao et al., Nat. Protoc. (2014)
Just out last week: Lattice-light sheet
(Eric Betzig’s lab, Janelia) Chen et al., Science (2014)
• One-photon excitation: high signal rate, multi-color, less cost
• Two-photon excitation: the gold standard for deep imaging
Excitation: pushing for the highest penetration depth
Zipfel & Webb (Cornell)
fluore
scence
excitation
fluore
scence
excitation
1-photon
excitation
2-photon
excitation
2-photon excitation: 940 nm
1-photon excitaton: 488 nm
• Longer wavelength penetrates
deeper due to less scattering
• Nonlinear confinement of signal
to “good” part of sheet
preservation of axial resolution
even if sheet is degraded
• Sheet thickness increases due to
scattering in heterogeneous sample
• Loss of axial resolution
as sample thickness increases
(even faster than lateral resolution)
Limits depth penetration
2-photon excitation to improves penetration depth of SPIM
Truong et al., Nature Methods (2011)
Truong et al., Nature Methods (2011)
4D imaging of entire embryonic development
of Drosophila (~ 1 day continuous imaging)
• Resolution: diffraction limited, subcellular
• Penetration depth: 2x better than 1p-SPIM; ≤ conventional 2p-LSM
• Imaging speed: >20x higher than conventional 2p-LSM
2p-SPIM simultaneously achieves high resolution, depth, speed
Truong et al., Nature Methods (2011)
Survey of latest developments in light sheet microscopy
• Excitation
• Detection
• Applications
Graphic from Huisken & Stanier, Development (2009)
Detection: How to improve the wide-field detection of SPIM?
• Deconvolution
• Multi-view imaging
+ joint deconvolution
• Confocal line detection of scanned sheet
with physical slit
with rolling shutter of sCMOS camera
Single views
Fusion Deconvolved fusions
Swoger et al., Optics Exp. (2007)
Baumgart et al.,
Optics Exp. (2012);
Imaging and Micro. (2013)
Survey of latest developments in light sheet microscopy
• Excitation
• Detection
• Applications
Graphic from Huisken & Stanier, Development (2009)
Survey of Applications: diverse and growing!
Developmental Biology
Imaging of endodermal migration in zebrafish embryo
(Huisken, MPI, Dresden)
Schmid et al., Nat. Comm. (2013)
Survey of Applications
Cell Biology
3D cell cultures
Observe fast intra-cellular processes
(Betzig, Janelia)
Chen et al., Science (2014)
Live imaging and analysis of spheroids
(Stelzer, Goethe Univ., Frankfurt)
Pampaloni et al., Cell Tissue Res. (2013)
Survey of Applications
Fixed/cleared large tissues
Imaging of entire mouse cochlea
Santi (Univ. Minnesota)
Buytaert et al., J. Histochem. & Cytochem. (2013)
Imaging of entire mouse brain
Deisseroth (Stanford)
Tomer et al., Nat. Protocol (2014)
Survey of Applications
Neuroscience
Whole-brain activity mapping in zebrafish larvae
Ahrens, Keller, Freeman (Janelia)
Vladimirov et al., Nat. Method. (2014)
Critical issue for SPIM applications: Sample Mounting
• Challenge: optical access for the side-illumination
• Now that the imaging is (mostly) non-toxic allows long observation
need novel mounting/handling to keep things alive
• Sample mounting/handling: “secret sauce” to successful SPIM application
*Agarose embedding often affects the biology*
www.zeiss.com
Chen et al., Science (2014)
Heart: first organ to form and function during development for humans: 3.5 weeks after conception Congential heart defects:most common birth defect 1% of births (40,000 infants per year) Heart disease is the leading cause of death in the US
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Application: Live Imaging of Vertebrate Heart with 2p-SPIM
Dynamic Mesoscopic regime connects structure & function
Physiological, Macroscopic scale
(~ dynamic)
Wik
iped
ia
The multi-scale challenge of studying the heart
Live embryonic zebrafish heart
MRI, Ultrasound, etc.
Structural, (Ultra-) Microscopic scale
(~ static)
Katz
, A
. M
. (2
001)
Physio
logy o
f th
e h
ea
rt.
Optical, Electron Microscopy
Zebrafish embryonic heart:
28
hpf
60 hours
post fertilization
(hpf)
scale bar
0.5mm
verterbrate, multi-chambered heart
share many genetic pathways with mammals
optical access
small size / sub-cellular ~ 500-um imaging
zebrafish, unlike mammals, can regenerate
their damaged heart
Use light sheet microcopy for 4D imaging of the live heart
To achieve:
subcellular resolution
over the entire 3D heart
over its beating cycle (cells moving ~150 um/sec)
20 μm
GT(tpm4-Citrine)Ct31a @ 84hpf,
fluorescence labels cardiomyocytes
Examples of Results: 2p-SPIM imaging and reconstruction of live zebrafish hearts
(Manuscript in preparation)
TgBAC(gata5:LifeAct-GFP) x Tg(gata1:dsRed)
@ 104 hpf
Green: cardiomyocytes; Red: blood cells
(Manuscript in preparation)
GT(ctnna-Citrine)Ct3a @ 84 hpf;
fluorescence labels cell boundaries between cardiomyocytes
(Manuscript in preparation)
Y Y
Light sheet microscopy
(orthogonal)
Conventional microscopy
(collinear)
• Parallelization high speed
• Detection cross-talk restricted to along focal plane only, not entire sample
• Low photodamage
- Light irradiates single focal plane at a time
- Laser excitation spread-out in space reduces peak laser intensity
Recap: Advantages of light sheet microscopy
* Willy Supatto (Polytechnique, Paris)
* Michael Liebling (UC Santa Barbara)
* Michel Bagnet (Duke)
* Principle Investigator: Scott Fraser
* The Fraser lab
* Heart project: Le Trinh, Vikas Trivedi
* SPIM: Cosimo Arnesano, John Choi,
Francesco Cutrale, Dan Holland,
Vikas Trivedi
Acknowledgement
NIH/NHGRI Grant # P50HR004071 NIH Grant #U01DE020063
Donna and Benjamin M. Rosen Center
Brought to you by the Science/AAAS Custom Publishing Office
Participating Experts
Thai Truong, Ph.D.
University of Southern California
Los Angeles, California
Pete Pitrone, DipRMS
MPI-CBG
Dresden, Germany
Webinar Series
Innovations in Light Sheet
Microscopy
Strategies and New Applications
October 29, 2014
Sponsored by:
Orla Hanrahan, Ph.D.
Andor Technology
Belfast, Ireland
Fast & Sensitive detectors for Light Sheet Microscopy Applications
55
How to choose the right camera
56
What makes a detector sensitive?
57
Two key parameters…
• Quantum Efficiency
• Noise floor
Camera must be designed to ensure these parameters are optimized.
0
0.1
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0.3
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0.5
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0.8
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1
300 400 500 600 700 800 900 1000 1100
sCMOS (4T)
sCMOS (5T)
Quantum Efficiency Curves
QE
Wavelength / nm
Shot Noise
Variation
Noise Floor
(Read Noise and Dark Noise)
Average
Signal Intensity
Making sense of sensitivity
• Read noise
‘Usual’ camera detection limit
• Dark noise
Dependent on temperature
• Shot Noise
QE and signal dependent
sCMOS – The detectors of choice for Light Sheet Microscopy
60
Scientific CMOS (sCMOS) is unique in
simultaneously offering:
• Extremely low noise
• Rapid frame rates
• Large field of view
• High resolution
• Wide dynamic range
Down to 0.9 e-
100 fps (full frame)
33,000:1
4.2/5.5 MegaPixel
4T sCMOS QE Advantage
QE
(%
)
Wavelength (nm)
4T sCMOS
5T sCMOS
sCMOS Exposure Modes
64
Special features on 4T sCMOS for applications of Light Sheet
Microscopy
65
Benefits: Image quality is improved since the scan row height can act as a slit detector, rejecting scattered light, improving contrast and SNR, therefore providing sharper and more resolved images.
Independent control over: 1. Pixel row Height (Slit Width) 2. Scan Speed 3. Exposure time
FlexiScan
66
Multiple readout modes
Benefit: Full chip can be used to scan the laser beam, rather than just one half
Enables maximum frame rate at full resolution Further control of the direction of readout on each half of the sensor Ideal for multi-wavelength applications
Thank you for your attention
67
Brought to you by the Science/AAAS Custom Publishing Office
Participating Experts
Webinar Series
Innovations in Light Sheet
Microscopy
Strategies and New Applications
October 29, 2014
Sponsored by:
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Thai Truong, Ph.D.
University of Southern California
Los Angeles, California
Pete Pitrone, DipRMS
MPI-CBG
Dresden, Germany
Orla Hanrahan, Ph.D.
Andor Technology
Belfast, Ireland
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Brought to you by the Science/AAAS Custom Publishing Office
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www.andor.com/light-sheet-microscopy
Sponsored by:
Innovations in Light Sheet
Microscopy
Strategies and New Applications
October 29, 2014