Biomedical Engineering Reference
In-Depth Information
microscope. utilizing. a. luorescent. bead. as. a. guide. star. [78]. embedded. in. a.
Drosophila
. embryo.. he.
specimen-induced. aberrations. were. measured. by. detecting. the. emitted. luorescence. with. a. SHWFS..
he ield.of.view.was.relatively.small.due.to.the.isoplanatic.angle.imposed.by.the.objective.used.
Another. study. using. an. SHWFS,. but. relying. on. backscattered. light,. has. recently. been. reported. by.
Denk.and.coworkers.[10,.79]..his.method.utilizes.the.backscattered.light.to.determine.the.aberrations..
Since.the.primary.interest.is.in.biological.tissues.that.are.highly.scattering,.a.few.issues.arise.with.the.use.
of.the.backscattered.excitation.light..he.irst.is.that.the.portion.of.the.backscattered.light.that.carries.
the.information.about.the.aberrations.in.the.focal.plane.forms.only.a.small.fraction.of.the.total.backscat-
tered.light..he.second.is.that.due.to.the.coherent.nature.of.the.backscattered.light.near.the.focus.and.
the.random.nature.of.the.scatterers,.speckle.is.formed..he.third.is.that.scattering.is.also.polarization-
dependent..To.address.these.three.issues,.Denk.and.coworkers.realized.that.the.most.important.back-
scattered.photons.to.be.collected.were.those.near.the.focus,.with.minimal.scattering..To.isolate.these.
photons,.a.coherence-gated.scheme.was.implemented.to.detect.only.the.ballistic.photons..he.issue.of.
speckle.was.addressed.by.averaging.the.speckle.through.slightly.moving.the.sample,.which.reduces.the.
speckle.contrast..It.is.also.known.that.in.a.relecting.sample,.such.as.the.retina,.coherent.light.picks.up.
aberrations.while.both.passing.into.and.out.of.the.eye.[68,.80]..he.result.is.that.odd.aberrations.are.lost.
because.they.are.replicated.in.the.return.pass.in.the.wrong.position,.while.even.aberrations.are.doubled,.
although.this.is.not.true.with.incoherent.light..To.overcome.this.issue,.Denk.and.coworkers.made.the.
assumption. that. even. though. the. backscattered. light. is. coherent,. the. random. nature. and. the. density.
of.the.distribution.of.the.scatterers.and.the.speckle.averaging.have.the.efect.of.rendering.the.detected.
backscattered.light.efectively.incoherent..his.meant.that.the.relected.(backscattered).light.was.efec-
tively.incoherent,.and.thus.it.carried.with.it.the.information.about.the.aberrations.only.on.the.return.
path,.thereby.making.an.SHWS.approach.possible.
More.recently,.Cha,.Ballesta,.and.So.[81].used.the.back-relected.light.from.the.sample.to.perform.AO.
multiphoton.microscopy..In.this.study,.Cha,.Ballesta,.and.So.used.a.two-photon.microscope.to.image.
the.brain,.heart,.and.tongue.of.a.mouse..A.pinhole.as.a.spatial.ilter.was.employed.before.the.SHWS,.
with. the. pinhole. size. being. just. above. the. size. that. would. transmit. the. maximum. spatial. frequency.
attainable.with.the.DM..his.method.is.an.alternative.to.using.an.interferometric.setup.for.coherence.
gating,. with. the. pinhole. efectively. accepting. only. the. light. from. near. the. focal. region.. In. this. work,.
aberration.correction.was.done.only.for.a.single.position.corresponding.to.the.center.of.the.image..In.
general,. adopting. a. single. coniguration. is. practically. useful. since. the. aberrations. present. are oten. a.
result. of. imaging. depth. (i.e.,. specimen-induced. or. refractive. index-induced. aberrations). [69, 82-84]..
In.this.study,.it.was.found.that.the.imaging.depth.was.typically.at.100-200.μm.and.the.signal.strength.
increased.on.the.order.of.20-70%..In.conclusion,.the.study.found.that.it.was.scattering.that.was.impor-
tant.in.determining.the.imaging.depth.as.it.degraded.the.image.on.an.exponential.scale,.whereas.deg-
radations.associated.with.aberrations.were.approximately.linear.
Up.till.this.point,.all.the.methods.have.been.based.on.expressing.the.aberrations.in.terms.of.Zernike.
or.Lukosz.modes..his.is.oten.done.because.of.their.orthogonality,.and.that.they.express.most.of.the.
.aberrations. very. well. and. can. be. easily. modeled. by. current. actuator-based. active.elements.. However,.
newer. active. elements,. such. as. the. SLM,. utilize. many. separate. and. individual. elements,. each. being.
independent.of.the.other..his,.of.course,.allows.for.a.departure.from.Zernike.or.Lukosz..polynomials..In.
a.recent.study.[85],.a.pupil-segmentation.approach.was.adopted.over.that.of.implementing.a..wavefront.
sensor.or.decomposition.into.orthogonal.modes,.such.as.Zernike.or.Lukosz.modes..Instead,.a..geometrical.
optics.approach.was.used,.where.the.pupil.of.the.objective.was.subdivided.into.smaller.subzones.that.
were. each. individually. corrected. using. phase. ramps. such. that. the. rays. focused. by. that. subzone. were.
shited. to. cross. at. the. focus.. In. general,. it. was. found. that. fewer. than. 100. subzones. were. required. to.
recover..difraction-limited.performance..his.was.achieved.via.two.methods—the.irst.being.a.“direct”.
measurement. method. in. which. a. reference. illumination. beam. (thin. pencil. of. rays). was. brought. to.
focus.along.with.another.beam.focused.along.a.small.subzone.of.the.pupil..he.variations.in.intensity.
(due. to. constructive. or. destructive. interference. between. the. reference. and. the. measurement beam).
