Biomedical Engineering Reference
In-Depth Information
Microscope
Microscope
Microscope
Wavefront
(a)
(b)
(c)
FIGuRE 13.2
Illustrations. depicting. (a). an. ideal. imaging. system,. (b). an. aberrated. imaging. system,. and. (c). an.
imaging. system. with. pre-compensation. for. sample-induced. aberrations.. he. thin,. sinusoidal. curves. denote. the.
phase.relationship.among.the.light.rays..(Ji,.N.,.et.al.,.
Nat. Meth
.,.7,.2,.141-147,.2010.)
indices.have.both.their.directions.and.phases.modiied.such.that.they.do.not.meet.at.a.common.spot.
and,.where.they.do.intersect,.they.interfere.less.constructively..Expressed.alternatively,.the.wavefront.
near.the.focal.region.is.no.longer.spherical,.but.rather.aberrated.(Figure.13.2b).
For.two-photon.luorescence.microscopy,.the.only.aberrations.afecting.the.image.quality.are.those.
experienced. by. the. focused. excitation. light,. since. the. luorescence. emission. is. detected. by. a. non-
image-forming.detector.and.does.not.convey.spatial.information..To.recover.the.difraction-limited.
resolution. and. the. optimal. signal,. we. need. to. modify. the. wavefront. of. the. excitation. light. before. it.
enters. the. objective,. so. that. each. ray,. even. ater. delection. and. phase. retardation. by. local. refractive.
index.inhomogeneities,.still.reaches.the.focal.point.at.the.optimal.phase.(Figure.13.2c)..To.accomplish.
this,.a.SLM.or.a.deformable.mirror.is.placed.along.the.excitation.light.path.at.a.plane.conjugate.to.the.
back. pupil. plane. of. the. objective. [9].. By. doing. so,. any. change. of. phase. at. a. given. point. on. the. SLM.
is. transferred. to. a. unique. corresponding. point. at. the. back. pupil. plane,. and. thus. on. the. excitation.
wavefront.
here.still.remains,.however,.the.issue.of.how.to.measure.the.delection.and.phase.ofset.of.each.ray.
without.a.wavefront.sensor..In.one.approach,.we.illuminated.only.one.segment.of.the.rear.pupil,.denoted.
as.the.“on”.subregion,.by.applying.a.lat.phase.to.the.corresponding.section.of.the.SLM.and.a.binary.
grating.of.phase.0.and.π.everywhere.else.(causing.the.light.in.these.regions.to.be.difracted.outside.the.
rear.pupil)..An.image.is.then.obtained.in.the.usual.manner.by.scanning.only.the.light.from.the.“on”.
subregion..If.the.light.ray.from.this.subregion.is.delected.by.refractive.index.inhomogeneities.along.its.
path,.the.luorescence.image.formed.thereby.is.shited.laterally.from.that.obtained.under.full-pupil.illu-
mination..From.the.magnitude.and.direction.of.this.shit,.the.two-dimensional.phase.gradient.needed.
to.shit.the.single-ray.image.back.to.the.position.of.the.full-pupil.one.is.calculated.and.then.added.to.the.
“on”.subregion.of.the.SLM..As.a.result,.the.original.ray.of.interest.is.tilted.such.that,.even.ater.traversing.
the.sample.inhomogeneities,.it.intersects.the.original.desired.focal.point..he.same.procedure.is.then.
repeated.for.other.subregions.of.the.SLM.till.the.complete.pupil.is.sampled,.and.all.rays.intersect.at.a.
common.point.(
Figure.13.3a-e
)
..Either.the.intrinsic.luorescence.in.the.sample.can.be.used.to.measure.
the.image.shit,.or.small.(0.1-2.0.μm).luorescent.beads.can.be.used.as.bright.and.photostable.“guide.
stars”.to.measure.the.shit.
Two. methods. can. then. be. used. to. determine. the. relative. phases. of. these. rays. that. maximize.
.constructive.interference..In.the.direct-measurement.approach,.one.reference.subregion.is.kept.on.at.all.
times,.and.each.of.the.other.subregions.is.turned.on.in.sequence..he.phase.of.each.of.these.other.rays.
is.varied.when.each.is.on,.until.the.interference.with.the.reference.ray.produces.maximum.intensity.at.
the.focal.point.(
Figure.13.3f-h
)..In.the.phase-reconstruction.approach,.the.phase.gradients.determined.
above.are.used.along.with.the.constraint.that.the.wavefront.is.continuous,.to.calculate.the.phase.algo-
rithmically.[10]..he.direct.approach.can.be.more.accurate,.since.an.error.in.one.pupil.subregion.does.
not.propagate.to.others,.whereas.the.phase-reconstruction.method.is.insensitive.to.photobleaching.and.
requires. relatively. few. luorescence. photons. for. accurate. measurement. as. well. as. substantially. fewer.
