Biomedical Engineering Reference
In-Depth Information
Bridgewater,. NJ). and. the. associated. single-photon. counting. circuitry.. he. scattered. light. signal. was.
relected. by. the. dichroic. scanner,. descanned. by. passing. through. the. scanning. mirrors,. and. directed.
toward.the.wavefront.camera.(HASO32;.Imagine.Optic)..he.wavefront.camera.was.also.positioned.at.a.
conjugate.plane.to.the.back.aperture.of.the.microscope.objective..A.confocal.pinhole.(P150S;.horlabs,.
Newton,.NJ).was.positioned.before.the.wavefront.camera,.and.at.a.plane.conjugate.to.the.focus.of.the.
objective.. his. completed. the. depth-discriminating. path. for. the. relected. light. signal.. As. mentioned.
earlier,. a. small. pinhole. is. necessary. for. good. axial. resolution.. However,. the. pinhole. needs. to. be. suf-
iciently.large.to.pass.adequate.information.about.the.distorted.wavefront.contained.in.the.spatial.fre-
quencies..In.this.particular.case,.the.minimum.pinhole.size.was.set.to.pass.the.highest.spatial.frequency.
that.was.obtainable.with.the.deformable.mirror..he.rationale.behind.this.was.that.a.pinhole.size.any.
larger.than.that.would.mean.that.the.deformable.mirror.could.not.correct.for.those.orders.of.aberra-
tions.while.any.smaller.size.would.mean.a.reduced.performance.of.the.deformable.mirror..he.distance.
between.any.two.actuators.on.the.deformable.mirror.is.2.5.mm.and.corresponds.to.a.minimum.spatial.
period.of.5.mm..A.Fourier-transform.of.this.spatial.period.gives.the.maximum.spatial.frequency.that.
needs.to.pass.through.the.wavefront-detection.light.path..We.determined.that.a.confocal.aperture.with.
a.diameter.no.less.than.125.μm.placed.at.plane.12.of.
Figure.16.1
.
would.transmit.all.the.relevant.aber-
ration.information..However,.we.chose.to.use.a.150-μm-diameter.pinhole.to.provide.some.additional.
margin.of.error..As.the.size.of.the.pinhole.essentially.afects.the.spatial.frequencies.passed,.and.this.in.
turn.is.inversely.proportional.to.the.depth.selectivity,.it.is.necessary.to.determine.the.axial.resolution.
aforded.by.this.150-μm-diameter.pinhole..Accounting.for.the.magniication.of.the.intermediate.optics,.
the. 150.μm. pinhole. at. plane. 12. corresponds. to. an. efective. aperture. of. 37.5.μm. radius. at. plane. 9. of.
Figure.16.1
.
.Since.the.focal.length.of.lens.10.is.125.mm,.the.efective.magniication.M.is.30.3..his.results.
in.an.axial.resolution.of.about.2.0.μm..his.resolution.is.deemed.suicient.because.the.back.aperture.of.
the.objective.is.underilled,.corresponding.to.an.efective.axial.resolution.of.4.8.μm,.and.is.discussed.
later..By.measuring.the.wavefront.distortion,.the.wavefront.camera.was.set.such.as.to.provide.feedback.
to.the.deformable.mirror..Depending.on.the.feedback.from.the.wavefront.sensor,.the.deformable.mir-
ror.generated.a.predistortion.to.the.incident.plane.wave.that.would.counter.the.distortion.induced.by.
the.sample..his.would.therefore.produce.a.PSF.at.the.focal.point.closer.to.the.ideal.and.improve.the.
two-photon.image.
An.important.issue,.as.described.by.Artal.et.al..[12],.is.now.addressed..It.is.to.be.noted.that.the.light.
detected. by. the. wavefront. camera. relects. of. the. deformable. mirror. twice,. while. the. actual. incom-
ing.excitation.light.to.a.sample.passed.the.deformable.mirror.only.once..Under.the.assumption.that.
the.specimen.is.highly.scattering,.this.is.a.reasonable.and.appropriate.assumption..In.the.irst.pass,.
the.light.is.aberrated..However,.all.phase.information.is.lost.on.difuse.scattering.and,.as.a.result, the.
backscattered. light. contains. information. only. about. the. aberrations. on. the. return/relected. path,.
with. the. aberrations. from. the. ingress. being. lost.. he. implication. of. this. is,. of. course,. that. such. an.
abberation-detection.scheme.in.the.transmission.will.pick.up.a.diferent.set.of.aberrations.and.is.not.
applicable.
Figures. 16.2
.
and.
16.3
.
show. representative. wavefronts. and. aberration. coeicients. before. and. ater.
AO.compensation..he.wavefront.sensor.provides.the.deformable.mirror.with.a.feedback.signal.while.
satisfying. of. the. Nyquist-Shannon. sampling. criterion.. he. AO. system. can. therefore. fully. utilize. the.
maximum. capability. of. the. deformable. mirror. for. wavefront. correction,. which. is. under. 10. nm. rms.
error,.according.to.speciications..At.the.same.time,.our.method.holds.only.when.the.sample.is.highly.
scattering.. his. is. oten. valid. for. biological. tissue. but. is. not. always. the. case.. For. samples. that. have.
closer approximate. specular. relection,. the. measured. wavefront. is. dependent. on. whether. the. aberra-
tions. generated. by. the. sample. are. odd. asymmetric. or. even. symmetric,. and. this. has. been. extensively.
discussed.[12-14]..In.terms.of.backscattered.light.eiciency,.the.majority.of.the.losses.are.due.to.the.use.
of.the.50:50.beamsplitter.for.the.excitation.and.detection.paths..hese.losses.can.be.avoided.by.using.a.
polarizing.beamsplitter,.followed.by.a.quarter-wave.plate.[15]..his.coniguration.relects.100%.of.the.
excitation.laser.power.instead.of.50%.in.the.absence.of.multiple.scattering.
