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Light sheet microscopy- Fast and gentle 3D imaging
Alessandro Ciccarelli –Light-sheet specialistCALM - Crick Advanced Light Microscopy Facility SW312
November 14, 2018
conversation
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• limited photon-budget (photobleaching)
• Doesn`t like light and fluorescence (photo-toxicity)
Imaging = conversation
E=hv !!!
E=hv ….
E=hv !!!
E=hv
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Light sheet microscopy
Wide field microscope+ illumination
• lateral• by a sheet of light
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Light sheet
Low photo-stress• 300 v < WF• 5000 v < LSM
Confocal microscopy Fast imaging
= WF100 > LSM
High SNRSNR 100v > than WF
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Amat et al., 2014
Drosophila Embryo
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McDole et al., 2018
Ms embryo, E 6.5 – E 8.5
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Figure 1
Belle et al., 2017
Human embryos –(Cleared )
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The Light-sheet microscope
Lasers
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“Build the microscope around the sample”Jan Huisken
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The light-sheets
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Focal plane
Light-sheet and spatial resolution • Lateral resolution = Wide Field + high contrast• Axial resolution depends on ratio Sheet/DOP
2x / NA 0.5
Depth of focus(DOP)
L-Sheet
Sheet < DOP:↑ Resolution
20 x / NA 1
Sheet > DOP:= Axial Resolution
↑↑↑ contrast
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Playing with Gaussian sheets
• NA of illumination lens determines the sheet thickness• The thinner in the center = the thicker on the borders
X
Z↑ NA
↓ NA
Focused Gaussian beam
• Image borders can be blurry• Need to adapt the sheet thickness to the sample• Stripes can happens
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The light- sheet journey
ImagingStorage Data
analysisSample
prep
Sample mounting
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Simaxglass
Agarose Beaker
Gentle and creative sample mounting
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Luxendo-BrukerMuVi-SPIM
Light-sheet @ The Crick
SW312 – LM4
1
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Camera Left
Camera Right
Back illumination
Front illumination
Photo mani pulator
zoom zoom
Temp control unit
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Detection
Illumination
Illumination
Detection
Sample chamber ( 8 ml)
w 20 X / NA 1
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Virtual (scanned) light-sheet
The MuVi-SPIM Light Sheet
X
Dual light-sheet
Light sheet diameter2,5 – 5,5 um
2,2 um
3,4 um
5,5 um
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Rolling shutter system
water
Agar +fluo beads
AREA mode Rolling shutter
Ovaries (Silvana G)
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Multicolor imaging with the MuVi Spim
405
445
488
515
594
642
418-462
457-501
497-554
499-569
LP 530
526-564
LP 498
LP 607
LP 656
610-651
Tomato
WL Band width
440 44
479 44
525.5 57
534 70
649 302
545 38
665 270
630.5 41
703.5 193
728 144
v20180625
Venus
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MuVi-SPIM Imaging
• Both left and right side are illuminated• Simultaneous detection of front and back side of the sample• Subcellular resolution
Camera Left Camera Right
Z Fish embryo (Andrew E)
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stack_0
stack_0
v 1
v 5
sample
MultiViewAcquistion
stack_1
stack_1
v 2
v 6
stack_2stack_2
v 3v 7
stack_3
stack_3
v 4
v 8
Z Fish embryo (Andrew E)
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8 views @ 45 deg
Z Fish embryo(Andrew E)
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MuVi-SPIM Resolution
Z-Proj (max int)
X
Y
Z
Z
Organoids (Jorge A)
Resolution Lateral = 300 nmAxial = 800 nm
Egg cgamber (Georgina F)
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Which sample (and how)
• Max sample size = 1 x 1 x 3 mm• Water based media (no clearing !!)• Live / Fixed
Glass capillary, ID: - 1 mm- 0,7 mm
FEP tubes (RI 1.33)
Detection lens
Illum
inat
ion
lens
sample
I
1 mm
FEP
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Dros. Embryo (Maxime T) Egg chamber (Georgina F)
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Dros. Pupa (Sophie H)
Ms Embryo (Clara C)
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Projects @ The Crick
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CAMP
- Store data (months)
10 Gb/s(real: 600 MB/s)
DATA WORKFLOW
wCAMP000
- Control MuVI-SPIM- Store data ( 1 day )- Quick analysis
InfinyBand*(40-50 Gb/s)
Cluster
Remote Desktop WORKSTATIONS
(CALM)
Lab PC
- Analysis
- Analysis
- Analysis
384 GB RAM
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H5 file
Imarismaster
IMARIS
Lux GUI
BDV
BDV
BigDataTools
H5 fileFUSED/resized
Load H5
• 3D Visualization
DATA PROCESSING
Elastik
FIJI
• Conversion• Opposite fusion• Multiview /rotation
fusion
• 2D Visualization• Processing• Stitching
BigSticher
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Samples• Small (< 1 mm)• Live (or fixed)
LuxendoMuVi-SPIM
• Embryos ( Zebrafish, Drosophila, Ms),• Organoids/spheroids • Blastocyst• Embryonic bodies• C Elegans?
- Cells tracking during development- Test drug effect on live sample- Understand 3D morphology of fragile sample- Ca2+ imaging- Optogenetics / photostimulation *
“ Gentle & live imaging ”
CO2/O2
- Cleared samples- Fluo intensity studies- 2D cell coltures- Confetti mice / spectral umixing
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2x / NA 0.5
Zoom0,63- 6,3 x
2
6 Sheets:5 – 12 um
sampleChamber: 120 ml
LavisionUltramicroscope II
Lens protection
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More even illumination (dynamic focus)
Sample size: 1 cm x 1 cm x 3 cm
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Laser lines and emission filters on the Lavision
488 561 638 705 785
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Autofluorescence
AlexaFluor 488Ex 488; Em 525/50
AlexaFluor 568Ex 561; Em 595/40
AlexaFluor 647Ex 640; Em 680/30
1 mm
Scattering
AbsorbtionRenier et al, Cell 2014
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Example of projects using the Lavision UMII
https://www.lavisionbiotec.com/products/UltraMicroscope/multiplexing.html
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Lavision test @ the Crick
Ms Bone (Stefania DB)
ECi Clearing
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LavisionUltramicroscope II
“Cellular question in large samples “
Samples• Fixed and large (1-10 mm)• Cleared (BABB, DBE, Eci,..)
- Vasculature, lungs alveoli,..- Find few cells in big organ- Tracing sparsely labelled neurons
• Ms brains• Ms embryos• Bones• Adult Drosophila
- Spine density- Count cells very densely packed - Live imaging- Cell colocalization
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• Young• New• Flexible
Low photo-bleaching/-toxicity FastGentle mounting
Summary - Light Sheets @ The Crick
size
Fixed
mm cm
MuVI
Live
UM II
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The light-sheet supporters
?? ?
?
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What`s for you
• Join the [email protected]
• Book training (PPMS) Muvi-SPIM Lavision UM *
• Write to me / Deborah Aubyn (CALM)
• Pop up at the facility (SW312)
• Join the Wednesday Imaging HelpDesk
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What`s next
- X-Clarity (Labtech):
- Seminar: Wed 21st November
- Demo: 3-7 December
- Intro to 2P microscopy (Rocco) Wed 28th 3 pm
- Luxendo Workshop: 13-15 February 2019
- Open SPIM afternoon
- Survey x users
- Any Q or suggestion?