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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


Microscopy


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


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)

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



• Excitation

• Detection

• Applications


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)


• Excitation

• Detection

• Applications


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)


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)


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

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ia

The multi-scale challenge of studying the heart

Live embryonic zebrafish heart

MRI, Ultrasound, etc.

Structural, (Ultra-) Microscopic scale

(~ static)

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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


GT(ctnna-Citrine)Ct3a @ 84 hpf;

fluorescence labels cell boundaries between cardiomyocytes


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



Thai Truong, Ph.D.




MPI-CBG

Dresden, Germany

Webinar Series


Microscopy


October 29, 2014

Sponsored by:


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

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

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



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October 29, 2014

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Thai Truong, Ph.D.




MPI-CBG

Dresden, Germany


Andor Technology

Belfast, Ireland

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