Biomedical Engineering Reference
In-Depth Information
his.implies. that.multiply. scattered.coherence-gated.light.has. penetrated. the.sample. less.deeply. than.
singly.scattered.light,.which.is.a.source.for.bias.in.the.wavefront.measurement.
In. addition,. the. decrease. in. speckle. size. implies. an. increase. in. speckle. error,. which. means. more.
interferograms.with.independent.scatterers.need.to.be.averaged.for.the.same.measurement.precision.
One.possible. solution. to.this.problem. could.be.the.use.of.an.aperture.in.a.plane.conjugated.to.the.
objective. lens. focus,. similar. to. the. pinhole. in. a. confocal. microscope.. Care. would,. however,. have. to.
be. taken. in. choosing. its. size;. a. subdifraction-sized. aperture. would. completely. ilter. all. aberrations,.
whereas. an. excessively. large. aperture. would. not. be. efective. in. iltering. out. the. out-of-focus. light..
Because.of.the.high.anisotropy.of.light.scattering.in.tissue.(most.scattering.events.change.the.light.angle.
by.only.a.small.amount),.most.multiply.scattered.light.passing.the.coherence.gate.can.be.expected.to.
take.paths.not.fundamentally.diferent.from.singly.scattered.light..his.implies.that.the.signal.that.is.to.
be.iltered.out.originates.in.direct.spatial.vicinity.to.the.useful.signal,.making.it.doubtful.if.a.suitable.
aperture.size.can.be.found.
14.6 critical Discussion
Coherence-gated.wavefront.sensing,.the.technique.reviewed.in.this.chapter.(Feierabend.2004;.Feierabend.
et.al..2004;.Rueckel.and.Denk.2005,.2006,.2007;.Rueckel.2006;.Rueckel.et.al..2006;.Tuohy.and.Podoleanu.
2010),.difers.from.most.other.current.techniques.in.wavefront.sensing.for.biological.microscopy.in.that.
it.does.not.rely.on.the.microscope's.regular.images.to.optimize.the.wavefront..Instead.of.analyzing.the.
images.taken.with.diferent.wavefront.bias,.simple.backscattered.light.is.used..For.luorescence.micros-
copy,.where.bleaching.is.oten.a.major.limitation,.this.has.the.advantage.that.no.luorescence.needs.to.
be.generated.during.wavefront.measurement,.so.the.full.luorescence.is.available.for.imaging.ater.the.
best.possible.wavefront.bias.has.been.determined..For.example,.although.2PM.needs.a.few.milliwatts.of.
average.laser.power.at.the.focus,.a.decrease.of.the.laser.power.by.three.orders.of.magnitude.would.still.
give.suicient.backscattering.to.measure.the.wavefront.using.CGWS.in.less.than.a.millisecond.
he.most.obvious.downside.of.CGWS.is.the.added.complexity.of.the.system,.since.an.interferometer.
needs.to.be.build.around.the.whole.microscope.
In.contrast.to.the.image-based.optimization.techniques,.CGWS.can.determine.all.aberration.modes.
from.the.same.dataset,.without.any.iteration..Since.no.bias.wavefronts.are.used,.sensitivity.to.certain.
modes.does.not.depend.on.the.ability.to.produce.such.a.mode..However,.it.should.be.noted.that.several.
sets.of.interferograms.need.to.be.taken.to.average.out.the.speckle.noise.that.is.unavoidable.in.interfero-
grams.because.of.the.coherent.backscattering.of.the.large.number.of.scatterers.in.the.CV.
To.correctly.measure.single-pass.aberrations,.CGWS.depends.on.scatterers.being.small.and.randomly.
distributed,.as.discussed.earlier,.in
.
Section.14.4
..he.assumptions.of.smallness.and.random.distribution.
seem.to.be.reasonably.well.fulilled.in.the.volume.of.many.biological.samples,.except.at.surfaces.and.
interfaces.between.regions.with.drastically.diferent.refractive.indices,.where.the.light.relected.by.the.
interface. could. cause. CGWS. to. measure. double-pass. aberrations. for. all. even. aberrations. modes. and.
zero.for.all.odd.aberration.modes..Variations.on.the.order.of.15%.in.the.slope.of.measured.Zernike.defo-
cus.are.seen.between.diferent.samples.(Rueckel.and.Denk.2007),.which.might.be.caused.by.violation.of.
the.scatterer.size.assumption,.but.more.research.is.needed.here.
he.current.implementation.using.phase.shiting.for.phase.retrieval.is.sensitive.to.sample.movements,.
which.can.cause.phase.steps.to.deviate.from.the.desired.value..his.issue.could.be.solved.by.replacing.
phase.shiting.with.a.phase.retrieval.scheme.based.on.of-axis.holography.(Leith.and.Upatnieks.1962),.
where.the.reference.wavefront.would.be.tilted.with.respect.to.the.sample.wavefront.
he. issue. of. multiple. scattering. decreasing. speckle. size. is. a. serious. one,. since. it. seems. inevitable.
at.higher.depths,.and.since.it.increases.speckle.error.for.a.given.number.of.interferograms..However,.
increasing.this.number.can.be.used.to.compensate.the.loss..Note.that.there.is.a.minimum.speckle.size.
because.of.the.inite.NA.of.the.transmission.optics.and.inite.wavelength;.it.is.unclear.whether.a.speckle.
pattern.with.this.minimum.speckle.size.can.still.carry.any.wavefront.information..For.CGWS.at.several.
