Biomedical Engineering Reference
In-Depth Information
We.have.developed.a.diferent.approach,.one.that.is.implemented.with.the.entire.pupil.illuminated.
at.all.times.and.thus.can.be.used.for.the.AO.correction.of.densely.labeled.samples.[11]..It.is.based.on.
the.same.simple.physical.picture.of.focal.formation—that.is,.an.ideal.focus.is.formed.with.all.light.rays.
converging.at.a.common.point.with.a.common.phase..Diferent.from.the.single-segment-illumination.
approach,.it.does.not.use.image.shit.to.determine.the.local.wavefront.tilt..Instead,.it.relies.on.the.fact.
that.a.light.ray.will.interfere.only.at.the.focus,.and.thus.modulate.the.focal.intensity.and,.consequently,.
the. signal. strength—most. efectively. when. it. intersects. the. focus. formed. by. all. the. other. light. rays..
hus,.scanning.one.ray.across.a.range.of.angles.while.keeping.all.the.others.ixed.would.vary.the.signal.
strength.(obtained.either.at.one.sample.position.by.parking.the.beam.or.as.the.averaged.signal.of.images.
by.scanning.the.beam)..Visualizing.the.signal.modulation.by.plotting.the.signal.relative.to.the.scanning.
ield.position.of.the.ray.as.an.image.(not.a.true.image.of.the.sample,.but.rather.a.map.relecting.the.varia-
tion.of.the.focal.intensity),.we.can.determine.the.local.wavefront.tilt..If.the.scanned.ray.faces.no.per-
turbing.inhomogeneities,.this.image.has.a.maximum.at.its.center,.corresponding.to.zero.applied.phase.
ramp.(Figure.13.4a.and.c)..Any.wavefront.tilt.causes.this.image.to.take.on.one.of.three.forms—an.image.
with.a.shited.maximum,.if.the.scanned.ray.constructively.interferes.with.the.ixed.rays.(Figure 13.4b.
and.d);.an.image.with.a.shited.minimum,.if.this.interference.is.destructive;.or.an.image.with.relatively.
lat.intensity,.if.the.phase.of.the.scanned.ray.relative.to.the.ixed.ones.is.near.±π/2..In.the.inal.case,.an.
additional.ofset.of.π/2.can.be.applied.to.the.scanned.ray,.and.the.measurement.repeated,.yielding.an.
image.with.a.shited.extremum..For.any.of.the.three.scenarios,.ray.delection—that.is,.local.wavefront.
tilt—can.be.measured.from.the.displacement.of.the.extremum,.just.as.in.a.Shack-Hartmann.sensor.or.
the.above.single-segment.illumination.algorithm..he.same.procedure.is.then.sequentially.applied.to.all.
rays/pupil-segments.across.the.entire.rear.pupil..Ater.all.the.wavefront.tilts.are.measured,.we.can.then.
determine. the. phase. of. each. ray. by. either. direct. measurement. or. phase. reconstruction,. as. described.
above..Because.this.approach.fully.illuminates.the.pupil.and.thus.utilizes.the.whole.NA.of.the.micro-
scope.objective,.it.can.be.applied.to.samples.of.arbitrary.3D.complexity.
It.is.worth.noting.here.that.even.though.both.approaches.we.outlined.here.are.zonal.AO.approaches.
and.both.rely.on.measuring.shits,.they.are.fundamentally.diferent..he.single-segment.illumination.
(a)
(b)
(c)
(d)
FIGuRE 13.4
Schematics. depicting. the. pupil-segmentation. method. with. full-pupil. illumination.. (a). An. ideal.
focus.has.all.rays.(arrowed.lines).intersect.at.the.same.point..(b).For.an.aberrated.focus,.rays.do.not.intersect.at.the.
same.point..Scanning.one.of.the.rays.(e.g.,.letmost.ray).through.a.range.of.angles.(shaded.cone).varies.the.intensity.
of.the.signal.excited.at.the.focus..(c).Plotted.as.an.image,.this.data.exhibits.an.intensity.extremum.centered.(dashed.
circle).over.the.scan.region,.if.the.ray.already.intersects.the.ideal.focus..(d).Shit.of.the.extremum.from.the.center.
indicates.local.wavefront.tilt..he.dashed.line.in.b.depicts.the.original.ray.direction,.while.the.solid.line.in.b.depicts.
the.direction.at.which.signal.extremum.is.reached..(Milkie,.D.E.,.et.al.,.
Opt. Lett
.,.36,.21,.4206-4208,.2011.)
