Biomedical Engineering Reference
In-Depth Information
(a)
(b)
(c)
(d)
FIGuRE 14.5
Comparing.the.light.propagation.and.backscattering.in.four.diferent.samples..For.clarity,.incom-
ing.waves.are.depicted.only.on.the.let.of.the.optical.axis.and.outgoing.waves.only.on.the.right.of.the.optical.axis..
Below.the.schematic.drawings.for.each.sample,.the.four.camera.images.taken.during.phase.shiting.are.shown,.as.
well.as.the.logarithm.of.the.amplitude.(middle.row).and.the.phase.(bottom.row).of.the.ield.reconstructed.by.phase.
shiting..Samples.are.(a).a.single.scatterer.(black.spot).in.the.focus.of.the.objective,.(b).an.axially.displaced.scatterer,.
(c).an.aberrating.layer.inside.the.sample,.and.(d).a.realistic.sample.
wave.is.transformed.by.the.objective.into.a.perfect.plane.wave.(Figure.14.5a)..Phase.shiting.will.reveal.
a.lat.phase.proile.in.the.BFP.(Figure.14.5,.bottom.row,.let).
For.a.scatterer.that.is.inside.the.CV.but.not.exactly.in.the.focus,.the.axial.displacement.will.cause.
the.wavefront.to.contain.piston.(absolute.phase).and.defocus*.(Figure.14.5b),.which.is.visible.as.a.radial.
phase.gradient.in.the.lower.row.of.Figure.14.5..A.lateral.displacement.of.the.scatterer.would.cause.the.
wavefront.to.be.tilted..Since.virtually.all.epidetecting.microscopic.techniques.are.insensitive.to.piston,.
tilt,.and.defocus.with.respect.to.signal.strength.and.resolution,.these.modes.do.not.have.to.be.corrected.
(apart.from.the.image.distortions.they.induce).
If.an.aberrating.layer.exists.between.the.objective.lens.and.the.point-like.scatterer,.already.the.incom-
ing.light.is.aberrated..his.causes.the.electric.ield.amplitude.of.the.incoming.light.at.the.position.of.the.
scatterer.to.be.decreased.(Figure.14.5c);.however,.since.the.scatterer.is.assumed.to.be.point-like,.the.spa-
tial.structure.of.the.aberrated.incoming.ield.does.not.change.the.spatial.structure.of.the.scattered.ield,.
which.is.still.spherical,.until.it.reaches.the.aberrating.layer..herefore,.the.wavefront.aberrations.that.
are.detected.in.the.BFP.correspond.directly.to.the.single-pass.aberrations.acquired.on.the.return.path..
If.the.density.of.scatterers.was.low.enough.to.have.only.one.scatterer.in.the.CV.at.the.same.time,.the.
phase.determined.by.phase.shiting.on.the.camera.would.correspond.directly.to.the.sample-introduced.
aberrations.that.need.to.be.corrected.by.adaptive.optics.to.provide.difraction-limited.focusing.in.the.
position.of.the.scatterer..In.Figure.14.5c,.this.is.shown.for.the.special.case.of.pure.astigmatism..Since.the.
*
.
It.should.be.stressed.that.defocus.in.high-NA.systems.does.not.correspond.directly.to.Zernike.defocus—it.is.not.simply.
a.quadratic.function.of.radial.position.in.the.BFP..Instead,.high-NA.defocus.contains.a.certain.amount.of.all.orders.of.
spherical.aberration..See.Botcherby.et.al..(2008).
