sted-spim microscopy « made simple
TRANSCRIPT
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STED-SPIM microscopy « made simple ».
Jean-Claude Vial; Teodora Scheul, Irène Wang Laboratoire LIPhy
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Aim : Combine SPIM and STED
• Why : to get thinner (but extended) light sheets
- Impossible with Gaussian beams
- Possible with Bessel beamsBut difficult to suppress peripheral light rings.
2
00
wZ
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STED
Able to give thinner fluorescent beams overpassing diffraction limits But need :
2 perfectly aligned beam.Perfectly synchronized lasers.A phase mask.
Need simplifications for a practical use
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STED-SPIM implementationA Leica patent in 2010Spim microscope with a sted light sheet US 20120098949 A1
A paper in 2011 by M.Friedrichet alSTED-SPIM: Stimulated Emission Depletion Improves Sheet Illumination Microscopy Resolution.
But only a modest (60%) light sheetthickness reduction is obtain
Need simplifications for a practical use
Two independant lasers, to be aligned, to be synchronized, a « classic » phase mask
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Simplification of the excitation & Stimulation source
Teem-photonicssub nanosecond
-Pump beam and STED beam obtained by harmonic generation :
-Beams are self aligned and self synchronized by design
DPSS Yag NdSelf Q-Switched A Single, bicolor
laser
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An excellent excitation-stimulation (depletion) source for blue dyes
400 500 600 700Abs
, Flu
o in
tens
ity (a
rb. U
n.)
Wavelength (nm)
532 nm355 nm
Coumarin 490 dye
Other favorable dyes:HoechstDAPINADH
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Highly efficient excitation- stimulation (depletion) source
0 5 10
Fluo
resc
ence
inte
nsity
(arb
. Un.
)
Distance from beam entrance (mm)0 5 10
Fluo
resc
ence
inte
nsity
(arb
. Un.
)
Distance from beam entrance (mm)
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An highly efficient excitation- stimulation (depletion) source
0.0 0.5 1.0 1.5 2.0 2.5 3.00.0
0.2
0.4
0.6
0.8
1.0
Nor
mal
ized
fluo
resc
ence
pro
file
z (mm)
PSTED= 33 µW PSTED= 181 µW PSTED= 700 µW
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.60
1
2
3
4
5
Average power at 532 nm (mW)
Low CW power
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The set up for STED-SPIMVery similar to the previous set up but need phase mask
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But need a dichroïc phase mask to preserve the co-alignment
4 segment mask made up with a dual wavelengthquartz waveplate
(Half wave at 532 nm and Wave at 355 nm)
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Sheet thickess reduction at modest power
Fluorescence beam section (µm)
220 µm
165
µm
0 2 4 6 8 10 12 14 16 18 20 22
0 2 4 6 8 10 12 14 16 18 20 22
7.4µm FWHM
2.1 µmFWHM
5.2 µmFWHM
1.8 µmFWHM
(a)
(b)
Fluorescence beam section (µm)
220 µm
165
µm
0 2 4 6 8 10 12 14 16 18 20 22
0 2 4 6 8 10 12 14 16 18 20 22
7.4µm FWHM
2.1 µmFWHM
5.2 µmFWHM
1.8 µmFWHM
(a)
(b)
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Sheets are more uniform
-120 -80 -40 0 40 800
2
4
6
8
10
Fluo
resc
ence
trac
e se
ctio
n (F
WH
M in
µm
)
Length of the fluorescence trace (µm)
Excitation alone
Excitation + Stimulation
Note : an initial Extension of the Rayleigh lengthDue to an importantSpherical aberration
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Sheet thickess versus STED power
0 5 10 15 200
1
2
3
4
(d0/d
)2
STED Power (kW/cm2)
Square of the reciprocal Of the sheet thickness The sheet thickness
Is reduced as the STED power increased
There is no limit in the size reduction
But ………
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Phase mask imperfections
0,0 2,5 5,0 7,5 10,0
Fluo
resc
ence
inte
nsity
(arb
.Un.
)
Fluorescence beam section (µm)
Increase of STED powerFrom 0 mw to 4 mW
The « zero » of light Is not perfect
4 quadrants are difficult to adjust
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Simplification of the dichroïc phase mask : 2 quadrants or less
Ultimate simplification : Just 1 quadrantA simple microscope coverslip(170 µm thickness). The index dispersion allows the dichroïc behavior The fine tuning is obtained by a small tilt.
Easilycentered
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Better efficiency of simplified masks
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Conclusion• It is possible to simplify the STED-SPIM
design• With a self Q-Switched subnanosecond laser• By harmonic generation of the pump and STED
beam Self aligned and self synchronized• With a dichroïc phase mask to preserve the co
alignment • With a 2 quadrant dichroic phase plate or a
simple coverslip.
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AcknowledgementsCoworkers : Téodora ScheulIrène Wang
Nanosciences foundation (PHD grant for Téodora)
ANR “Nanoscolas grant”
Teem Photonics (Laser loan)