Biomedical Engineering Reference
In-Depth Information
ND2
M3
Reference arm
M5
BSC2
RP
GVD-P
M4
M7
TS2
Calibration arm
M6
PZT
DM
PMT
L3
L4
pBSC
M8
M9
CCD
L7
F
BSC1
λ /2
M Dichr.
M2
Pump
laser
λ /4
Ti:Sa
M10
L5
L6
M1
Sample arm
OBJ
ND1
L1
L2
T
TS1a
Computer
TS1b
FIGuRE 14.8 CGWS.and.DM.integrated.into.a.two-photon.microscope..From.Rueckel,.M.,.J..A..Mack-Bucher,.
et  al.. (2006).. Adaptive. wavefront. correction. in. two-photon. microscopy. using. coherence-gated. wavefront. sensing..
Proc. Natl. Acad. Sci. USA . 103 (46):17137-17142..Copyright.2006.National.Academy.of.Sciences,.USA. 
Two-photon.microscopy.is.similar.to.confocal.microscopy.in.that.it.is.a.luorescence-based.laser.scan-
ning.microscopy.providing.three-dimensional.resolution..In.contrast.to.confocal.microscopy,.2PM.does.
not.rely.on.the.luorescent.light.from.the.focus.reaching.a.conjugated.focal.point.on.a.ballistic.trajectory,.
where.it.can.be.spatially.iltered.for. z -selectivity..Instead,.two.excitation.photons.with.half.the.energy.(and.
hence.twice.the.wavelength).are.absorbed.in.one.quantum.event..his.two-photon.absorption.process.has.
a. probability. that. depends. quadratically. on. the. excitation. power,. providing. z -selectivity. already. at. the.
level.of.absorption..he.fact.that.this.allows.to.detect.and.correctly.assign.luorescent.photons,.even.if.they.
have.been.scattered.on.their.way.toward.the.detector,.together.with.the.decrease.in.scattering.of.the.excita-
tion.light.associated.with.the.doubling.of.the.wavelength,.explains.the.superior.penetration.depth.of.2PM,.
which.has.been.shown.to.be.feasible.down.to.depths.of.1.mm.in.cortical.tissue.(heer.and.Denk.2006).
To.reach.the.high.instantaneous.excitation.powers.necessary.for.two-photon.excitation.while.keep-
ing.the.average.power.low,.femtosecond.laser.pulses.are.usually.used..In.our.experiments,.a.titanium-.
sapphire.(Ti:Sa).laser.operating.at.930.nm.was.used..Since.the.spectral.width.of.the.laser.pulses.had.a.
full.width.at.half.maximum.(FWHM).of.15.nm,.the.coherence.length.of.the.two-photon.laser.was.short.
enough.to.make.it.a.feasible.source.for.CGWS,.resulting.in.an.FWHM.of.the.CV.of.19.μm..his.has.the.
advantage.that.the.same.source.can.be.used.for.both.wavefront.measurement.and.imaging.
he.laser.beam.from.the.Ti:Sa.is.attenuated.with.a.neutral.density.ilter.(ND1).and.its.size.increased.
with.a.telescope.(L1/L2).before.it.enters.the.interferometer..To.adapt.the.proportion.of.the.light.entering.
the.two.arms.of.the.interferometer,.a.polarizing.beam.splitter.cube.(pBSC).is.used.in.conjunction.with.a.
half-wave.plate.(λ/2).that.rotates.the.linear.polarization.of.the.incoming.laser.beam.
Compared.to.the.simpliied.CGWS.setup.discussed.earlier,.the.sample.arm.(to.the.right.of.the.pBSC).
becomes.much.longer.since.the.DM.used.for.wavefront.correction.needs.to.be.conjugated.to.the.objec-
tive.lens.BFP..One.telescope.(L3/L4).corresponding.to.the.telescope.in. Figure.14.4 .conjugates.the.CCD.
camera.with.the.DM,.while.a.second.telescope.(L5/L6).is.necessary.to.conjugate.the.DM.to.the.BFP.of.
the.objective.lens.(OBJ).
As.a.result.of.the.increased.arm.length.dictated.by.the.two.telescopes,.the.reference.arm.(upper.half.
of.Figure.14.8).is.folded.using.a.large.number.of.mirrors.to.make.it.it.onto.the.optical.table..To.match.
 
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