Biomedical Engineering Reference
In-Depth Information
approach.relies.on.measuring.the.shit.of.sample.images,.conceptually.similar.to.the.principles.underly-
ing.a.Shack-Hartmann.sensor,.while.the.full-pupil.illumination.approach.relies.on.the.intensity.modu-
lation.of.the.focus,.independent.of.image.contrast.mechanism.and.image.structure..In.the.13.4,.we.will.
show.how.both.these.approaches.can.recover.difraction-limited.resolution.through.highly.aberrating.
samples.
13.3 experimental Setup
Even. though. our. approach. can. be. easily. adapted. to. other. point-scanning. modalities,. such. as. those.
relying.on.second-harmonic.generation,.third-harmonic.generation,.and.Raman.scattering,.we.imple-
mented.the.pupil-segmentation-based.AO.approach.in.a.two-photon.luorescence.microscope.because.
of. its. widespread. usage. in. bioimaging. (Figure. 13.5).. Near-infrared. femtosecond. pulses. generated. by.
a. titanium-sapphire. laser. (Chameleon. Ultra. II;. Coherent. Inc.). are. raster-scanned. in. 2D. by. a. pair. of.
.galvanometers.(X.and.Y).(6215H;.Cambridge.Technology.Inc.)..he.galvanometers.are.optically.conju-
gate.to.each.other.with.two.custom-made.30.mm.focal-length.telecentric.f-θ.lenses.(F1).(Special.Optics)..
A.third.F1.lens.and.a.custom-made.150.mm.focal-length.telecentric.f-θ.(F5).lens.(Special.Optics).conju-
gate.the.Y.galvanometer.to.a.liquid-crystal.phase-only.SLM.(1,920.×.1,080.pixels;.PLUTO-NIR;.Holoeye.
Photonics.AG)..he.conjugation.of.the.galvanometers.to.the.SLM.ensures.that.the.intensity.at.each.sub-
region.of.the.SLM.remains.constant.during.beam.scanning..he.SLM.is.itself.either.conjugated.by.a.pair.
of.F5.lenses.to.the.rear.pupil.of.a.20.×,.NA.1.0.water-dipping.objective.(W.Plan-Apochromat;.Carl.Zeiss.
Inc.),.or.by.a.custom-made.120.mm.focal-length.telecentric.f-θ.(F4).lens.and.a.custom-made.240.mm.
focal-length.telecentric.f-θ.(F8).lens.(Special.Optics).to.the.rear.pupil.of.a.16.×,.NA.0.8.water-dipping.
objective.(LWD.16.×.W;.Nikon.Corp.)..For.the.underilled.Zeiss.objective,.the.SLM.area.used.in.AO.cor-
rection.is.rectangular,.whereas.for.the.overilled.Nikon.objective,.the.SLM.area.is.square..Conjugation.
of. the. SLM. to. the. objective. rear. pupil. ensures. that. the. corrective. phase. pattern. applied. at. the. SLM.
would.be.transferred.to.the.rear.pupil.and.remain.stationary.during.beam.scanning..he.objective.is.
mounted.to.a.piezo-lexure.stage.(P-733.ZCL;.Physik.Instrumente,.GmbH).for.2D.and.3D.imaging.in.
the.axial.direction..he.luorescence.signal.is.split.into.red.and.green.components.and.detected.by.two.
M1
X
Y
FLR
BS
TPE
BS
L2
FL2
F5(F8)
F1
F1
F1
"3$
"3$
PMT
"
PD
"#
L1
EOM
FW
FS
ZP
Obj
L3
Ti:Sa
$%&'(
S
LP
BP
BE
FL1
BR
F5
"3$
"3$
PMT
F5(F4)
SLM
FIGuRE 13.5
Schematic. of. an. AO. two-photon. luorescence. microscope.. From. let. to. right:. Ti:. Sapphire. laser.
(Ti:Sa);.electro-optical.modulator.(EOM);.2×.beam.expander.(BE),.beam.pickof.(BP).that.relects.~3%.of.the.light.
into. a. photodiode. (PD). and. a. neutral-density. ilter. wheel. (FW). for. excitation. power. control;. motorized. beam.
reducer. (BR);. two-axis. laser. positioner. (LP);. X. galvanometer. (X);. two. 30. mm. focal-length. telecentric.
f
-θ. lenses.
(F1);.Y.galvanometer.(Y);.30.mm.(F1).and.150.mm.(F5).focal-length.telecentric.
f
-θ.lenses;.spatial.light.modulator.
(SLM);.two.150.mm.focal-length.lenses.(F5).for.Zeiss.objective,.one.120.mm.(F4).and.one.240.mm.(F8).lens.for.
Nikon.objective;.ield.stop.(FS);.mirror.(M1);.dichroic.beamsplitter.(TPE.BS);.Zeiss.or.Nikon.water-dipping.objec-
tive.(Obj).mounted.on.a.Z-piezo.stage.(ZP);.sample.(S);.luorescence.collimating.lens.(L1);.dichroic.beamsplitter.
(FLR.BS).to.separate.green.and.red.luorescence;.two.lenses.(L2,.L3).to.focus.luorescence.through.two.ilter.sets.
(FL1,.FL2).onto.two.photomultiplier.tubes.(PMT)..(Ji,.N.,.et.al.,.
Nat. Meth
.,.7,.2,.141-147,.2010.)
