Biomedical Engineering Reference
In-Depth Information
16.4 conclusions
Wavefront.distortion.by.tissue.specimens.with.inhomogeneous.optical.properties.can.be.measured.by.
an.SHWS.using.a.confocal.depth.selection.mechanism..he.measured.wavefront.distortion.can.then.be.
corrected.by.a.deformable.mirror.integrated.into.the.microscope.system..In.specimens.with.large.aber-
ration,.such.as.artiicial.specimens,.signiicant.improvement.in.signal.strength.and.resolution.can.be.
achieved..In.more.realistic.tissue.specimens,.the.imaging.depth.is.limited.to.100-200.μm.by.the.respec-
tive. scattering. coeicients. of. the. respective. tissue. type.. For. example,. in. general,. achievable. imaging.
depth.for.the.brain.tissue.is.approximately.three.times.that.of.the.muscles..he.measured.aberration.for.
these.tissues.at.this.depth.is.similar.to.those.reported.by.other.investigators.[8,20]..he.corresponding.
improvement.in.terms.of.signal.strength.is.on.the.order.of.20-70%..However,.the.improvement.on.the.
peak.signals.(90-100%.strength.intensity.signals).is.up.to.twofold..his.means.the.contrast.of.images.
was.increased.ater.AO.compensation;.therefore,.the.images.became.more.vivid,.and.this.is.one.of.the.
most.essential.advantages.of.the.AO.system.in.biological.imaging..Our.data.appears.to.imply.two.related.
conclusions..First,.tissue.scattering.is.the.primary.factor.that.determines.the.maximum.imaging.depth.
in.tissue.imaging.by.a.two-photon.microscope..While.the.efect.of.scattering.is.exponential.with.depth,.
the.accumulated.aberration.as.a.function.of.depth.appears.to.remain.at.the.same.order.of.magnitude.
or.varies.at.the.most.linearly..herefore,.the.scattering.efect.is.always.the.domi.nant.factor..Second,.AO.
control.can.improve.the.imaging.signal.at.depth..However,.typical.improvement.is.relatively.modest,.
especially.for.highly.scattering.specimens,.because.the.very.limited.imaging.depth.implies.relative.little.
accumulated.aberration,.limiting.the.improvement.that.can.be.obtained.by.using.AO.correction..hese.
conclusions. are. consistent. with. most. of. the. AO-compensated. nonlinear. optical. imaging. studies.. he.
only.exceptions.are.the.application.of.AO.compensation.in.CARS.microscopes..One.potential.explana-
tion.may.be.that.the.CARS.signal.is.highly.dependent.on.the.overlap.of.the.excitation.volumes.of.the.
pump.and.probe.beams,.and.aberration.may.result.in.spatial.mismatch..he.correction.for.aberration.
can.optimize.volume.overlap.and.result.in.the.observed.sixfold.improvement.[5]..It.might.therefore.be.
important.to.ask.if.AO.correction.is.important.for.two-photon.microscopy..Our.data.seem.to.indicate.
that. for. most. tissue. types. with. high. scattering-coeicient. and. shallow. imaging-depth,. adaptive. cor-
rection.can.improve.the.signal.strength..However,.one.should.note.that.if.the.excitation.power.is.not.
limited.and.photodamage.is.not.an.issue,.this.level.of.improvement.can.be.equivalently.achieved.by.less.
than.a.twofold.excitation.power.increase..In.the.case.that.higher.excitation.power.cannot.be.applied,.AO.
can.provide.some.improvement,.although.at.the.expense.of.an.increase.in.instrument.complexity.and.
cost..It.may.also.be.useful.to.consider.in.what.situations.AO.correction.would.be.useful.for.two-photon.
imaging.. Clearly,. in. tissues. with. relatively. low. scattering-coeicients. that. allow. deep. imaging,. AO. is.
useful.to.correct.for.the.larger.aberration.present..his.is,.of.course,.the.well-known.case.with.the.clas-
sic.examples.of.imaging.in.organs,.such.as.the.eye.[21-23]..Other.potential.application.areas.are.tissues.
that.induce.unusually.high.aberrations..Another.classic.example.is.the.skin,.where.the.stratiied.layers.
have.very.diferent.indices.of.refraction.and.result.in.signiicant.aberration,.even.in.relatively.shallow.
depths..It.would.be.interesting.to.examine.the.possible.application.of.AO.compensation.in.skin.imag-
ing..In.addition,.there.are.several.other.potentially.interesting.applications.of.AO.in.nonlinear.micros-
copy.imaging.of.tissues..First,.tissue.scattering,.due.to.a.combination.of.Rayleigh.and.Mie.processes,.
is.lower.for.light.at.longer.wavelength.regions,.which.corresponds.to.deeper.penetration.depth..If.the.
AO.system.is.combined.with.a.two-photon.microscope.operating.in.the.1.2-1.3.μm.region.and.imaging.
near-infrared. emitting. luorophores,. one. may. achieve. deeper. imaging. in. tissues. with. corresponding.
higher.aberration.efect..In.this.case,.one.can.expect.AO.compensation.to.be.more.important..Second,.if.
the.scattering.efect.becomes.negligible,.then.the.aberration.due.to.the.inhomogeneous.refractive.index.
of. a. sample. becomes. the. major. factor. for. image. degradation.. One. obvious. example. is. in. specimens.
with. extensive. optical. clearing. that. can. dramatically. decrease. tissue. scattering. by. dehydration. of. the.
specimen.and.the.addition.of.an.index.matching.medium.[24,25]..Optical.clearing.reduces.scattering.
by. index-matching. nanometer-scale-level. inhomogeneity. in. the. specimen.. With. the. optical. clearing,.
