sted-spim microscopy « made simple
TRANSCRIPT
STED-SPIM microscopy « made simple ».
Jean-Claude Vial; Teodora Scheul, Irène Wang Laboratoire LIPhy
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
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
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
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
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
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)
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
The set up for STED-SPIMVery similar to the previous set up but need phase mask
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)
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)
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
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 ………
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
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
Better efficiency of simplified masks
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.
AcknowledgementsCoworkers : Téodora ScheulIrène Wang
Nanosciences foundation (PHD grant for Téodora)
ANR “Nanoscolas grant”
Teem Photonics (Laser loan)