Biomedical Engineering Reference
In-Depth Information
When.using.luorescent.microspheres.for.guide.stars,.the.microsphere.solution.concentration.injected.
into.the.embryo.ensures.that.there.is.at.least.1.microsphere.within.10.μm.of.the.injection.site..his.can.
be.used.to.accurately.target.diferent.embryo.locations.for.imaging..Higher.microsphere.concentrations.
have.also.been.tested.and.the.results.showed.that.beads.can.spread.much.more.densely.without.impact-
ing.embryo.development..Having.multiple.microspheres.relatively.close.to.each.other.does.not.present.
a.problem.since.the.confocal.illumination.setup.used.in.this.experiment.can.accurately.target.one.bead.
at. a. time.. Experimental. results. showed. that. even. if. two. or. more. microspheres. are. in. the. same. focal.
plane.and.are.“relatively”.close.to.each.other.(i.e.,.within.5.PSF's.FWHM),.the.wavefront.sensor.would.
see.an.extended.object.and.the.resulting.measurement.would.be.the.average.wavefront.seen.from.each.
microsphere.(Azucena.et.al..2010)..Microspheres.that.are.in.diferent.focal.planes.present.even.less.of.a.
problem.since.the.inite.focus.of.the.confocal.illumination.prevents.them.from.being.fully.illuminated.
(because.of.the.high.NA),.and.hence,.they.are.not.the.brightest.object.in.the.FOV..Microspheres.that.are.
in.diferent.focal.planes.do.show.up.as.a.background.light.in.the.wavefront.sensor.camera..It.has.been.
shown.by.homas.et.al..(2006).that.a.robust.centroiding.algorithm,.like.the.cross-correlation.technique.
used. here,. does. not. sense. the. background. and. detects. only. the. brightest. object. in. the. FOV,. be. it. an.
extended.or.a.point.source..his.still.holds.true.even.for.a.very.small.SNR.(i.e.,.the.background.would.
add.to.the.peak.of.the.Hartmann.spot.centroid.but.it.does.not.move.it)..he.same.can.be.said.for.light.
that.is.being.scattered.inside.the.embryo..It.adds.to.the.background.but.it.does.not.change.the.wavefront.
sensor.measurement.
he.accuracy.and.stability.of.direct.wavefront.sensing.depends.on.the.amount.of.light.from.the.luo-
rescent.microsphere..he.lateral.and.axial.positions.of.the.microsphere.afect.the.accuracy.of.the.wave-
front.measurement..In.astronomy,.the.isoplanatic.angle.was.deined.as.a.relative.measure.of.the.FOV.
over.which.the.AO.system.can.operate..In.AO.microscopy,.the.isoplanatic.half-width.was.used.as.the.
size.of.the.FOV.that.the.AO.system.can.correct..It.has.been.tested.for.the.
Drosophila
.embryo.using.a.40×.
objective.(0.75.NA)..his.value.varies.for.diferent.types.of.samples.and.objectives..It.will.be.evaluated.
for.mouse-brain.tissue.in.future.research..In.the.confocal.microscope,.the.images.are.acquired.point-
by-point.and.reconstructed.by.the.computer..If.there.are.multiple.microspheres.in.the.FOV,.AOCFM.has.
the.ability.to.correct.the.diferent.aberrations.for.diferent.parts.of.the.FOV.in.one.image..he.variation.
of.wavefront.error.along.with.the.depth.is.also.being.investigated..Although.the.wavefront.measurement.
requires.microspheres.in.the.focal.plane,.the.wavefront.can.also.be.estimated.by.linear.interpolation.if.
measurements.of.the.wavefront.are.available.in.at.least.two.planes.close.to.the.focal.plane.
For.a.live.sample,.real-time.wavefront.measurement.and.correction.is.a.more.challenging.task..he.
wavefront.measurement.is.based.on.the.emission.light.from.the.microsphere..However,.the.internal.low.
or.motion.of.the.liquid.inside.the.cell.or.tissues.can.cause.an.unpredictable.motion.of.the.microsphere..
Focusing.the.illumination.laser.onto.the.microsphere.in.three.dimensions.can.also.be.diicult..One.pos-
sible.solution.is.to.steer.the.beam.to.compensate.for.the.motion.of.the.microsphere.with.an.appropriate.
image.stabilization.algorithm.
An. emerging. ield. in. AO. is. tomography. AO,. where. multiple. light. sources. together. with. multiple.
SHWF.sensors.are.used..he.information.from.each.wavefront.sensor.is.then.processed.using.a.recon-
structor. to. acquire. a. tomographic. image. of. the. changes. in. the. refraction. index. in. the. optical. path.
(Hardy,. 1998).. One. of. the. advantages. of. using. tomography. AO. is. that. it. can. provide. information. on.
the.depth.dependence.of.variations.in.the.refraction.index.in.the.tissue,.thus.allowing.the.AO.system.
to. correct. for. the. wavefront. aberrations. only. in. the. optical. path.. his. technique. can. also. extend. the.
isoplanatic.angle.by.correcting.wavefront.aberrations.that.are.common.to.a.larger.FOV..By.depositing.
multiple.luorescent.beads.into.the.biological.sample.and.using.multiple.wavefront.sensors,.we.can.also.
apply.the.tomographic.techniques.that.have.been.developed.for.astronomical.AO.
AO.wide-ield.and.confocal.microscopes.were.designed.using.an.SHWF.sensor.and.Micro-Electro-
Mechanical.Systems.DM.to.directly.measure.and.correct.the.wavefront.error.induced.by.the.biologi-
cal. samples.. For. the. wide-ield. microscope,. the. wavefront. measurements. were. taken. by. using. a. new.
