Biomedical Engineering Reference
In-Depth Information
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FIGuRE 15.26
Example.phase.diversity.on.a.luorescence.image..(a).In-focus.image.of.GFP-labeled.
Arabidopsis
..
(b).Out-of-focus.image..(c).Object.estimated.from.phase.diversity..(d).Wavefront.estimated.from.phase.diversity.
(radians)..(e).Axial.cross.section.of.image.stack.showing.the.efect.of.the.spherical.aberration..Scale.bars.are.5.μm.
A.potential.advantage.of.phase.diversity,.as.with.phase.retrieval,.over.other.wavefront.sensorless.tech-
niques.is.that.it.could.potentially.require.fewer.images.to.determine.the.wavefront.aberration..Sensorless.
techniques.typically.require.many.images.to.optimize.the.wavefront..he.number.of.images.required.
typically.depends.on.the.number.of.degrees.of.freedom.to.be.optimized..Phase.diversity.can.in.principle.
determine.the.full.wavefront.from.only.two.images.
Figure.15.26.shows.the.use.of.phase.diversity.on.a.thin.sample..An.in-focus.and.an.out-of-focus.image.
of.a.thin.sample.from.an.
Arabidopsis thaliana
.stem.in.which.the.cell.walls.are.labeled.with.Alexa.488.
are. used. as. the. input. to. the. phase-diversity. algorithm,. and. the. wavefront,. parameterized. by. the. irst.
15 Zernike.modes,.that.best.reproduces.the.images.is.determined..hese.images.were.taken.using.a.40×.
air. objective. with. a. correction. collar. and. show. spherical. aberration. because. of. the. setting. of. the. col-
lar..he.calculated.wavefront.(Figure.15.26d).correctly.captures.the.spherical.aberration.present.in.the.
images..Figure.15.26e.shows.an.axial.cross.section.of.the.image,.showing.the.asymmetry.in.the.out-of-
focus.light.that.is.characteristic.of.spherical.aberrations.
15.4 conclusions
We.have.discussed.our.work.on.using.AO.to.correct.aberrations.in.wide-ield.luorescence.microscopy..
To. correct. only. depth. aberrations,. it. is. possible. to. use. an. open-loop. approach,. where. the. mirror. can.
be. set. based. on. a. priori. knowledge. of. the. depth. and. sample. refractive. index.. For. more. complicated.
samples,.where.sample-induced.aberrations.are.signiicant,.this.approach.is.not.suicient..We.show.that.
phase.retrieval.on.a.luorescent.bead.can.be.used.to.correct.even.severe.aberrations,.and.we.discuss.the.
possibility.of.using.phase.diversity.to.eiciently.measure.aberrations.in.biological.samples.
AO.promises.to.have.a.signiicant.impact.on.wide-ield.luorescence.microscopy,.but.there.is.much.
work.to.do.before.its.use.is.routine..he.diferent.approaches.to.measuring.and.correcting.the.wavefront.
must.be.evaluated.and.optimized.before.images.can.be.reliably.taken.with.one.click.of.the.mouse..here.
are.also.many.areas.of.development.for.AO.in.wide-ield.microscopy.
