Biomedical Engineering Reference
In-Depth Information
around.slightly,.and.that.the.midpoint.between.the.scanners.is.reimaged.onto.the.back.aperture.of.the.
microscope.objective.rather.than.onto.a.true.stationary.scan.point..For.a.reimaging.system,.one.scan-
ning.mirror.is.relayed.onto.the.second,.using.either.lenses.or.mirrors.with.the.inal.mirror,.then.is.reim-
aged.onto.the.back.aperture.of.the.microscope.objective,.ensuring.that.one.has.a.true.stationary.point.
in.the.back.plane..Clearly,.this.involves.more.optical.components.but.theoretically.it.provides.a.better.
imaging.system..In.practice,.when.real.biological.samples.are.placed.on.the.sample.stage.both.conigu-
rations.work.well.and.are.used.in.accepted.commercial.microscopes.
As.mentioned.earlier,.the.scanned.beam.now.has.to.be.relayed.onto.the.back.aperture.of.the.objective.
such.that.the.beam.pivots.around.the.aperture.rather.than.being.spatially.scanned..However,.there.is.a.
further.consideration..To.exploit.the.full.numerical.aperture.of.the.objective.lens,.the.beam.needs.to.ill.
the. back. aperture,. which. will. vary. from. objective. to. objective.. Typically,. systems. are. optimized. for. the.
largest.back.aperture.and.for.lenses.with.smaller.apertures.the.user.just.accepts.the.light.loss..he.beam.
expansion.required.is.normally.around.10.times.so.that.the.1.mm.diameter.laser.beam.then.ills.the.10.mm.
diameter.objective..Again,.this.relaying.and.expansion.can.be.achieved.either.using.lenses.or.mirrors.with.
the.advantage.of.mirrors.being.their.achromatic.performance.
In.nonlinear.microscopes,.as.mentioned.earlier,.the.detector.does.not.have.to.be.mounted.such.that.
the.emission.beam.is.descanned.before.reaching.the.detector.as.the.nonlinear.excitation.provides.inher-
ent. optical. sectioning. negating. the. need. for. a. confocal. pinhole. for. three-dimensional. imaging.. he.
detector.can.thus.be.placed.in.any.of.the.three.positions.as.illustrated.in.
Figure.11.1
..In.the.case.of.CARS.
and. harmonic. imaging,. which. are. coherent. processes,. the. natural. direction. of. the. signal. light. is. for-
ward.rather.than.being.isotropic.in.the.case.of.luorescence-based.imaging..his.means.that.a.forward-.
mounted.detector.frequently.provides.the.largest.signal.even.if.it.is.inconvenient.to.mount..he.entire.
system.is.clearly.controlled.by.computer,.which.operates.the.scanning.mirrors,.the.light.collection,.and.
the.Z-drive.on.the.microscope.
11.2.2 incorporation of the Adaptive optic element: Pre- and Postscanning
We.now.need.to.consider.the.insertion.of.the.AO.element.and.there.are.two.options:.before.or.ater.the.
scanning.optics..In.both.conigurations,.the.adaptive.element.needs.to.be.reimaged.to.ensure.that the AO.
element,.scanner,.and.back.aperture.are.all.conjugate.with.each.other..If.this.is.not.achieved,.the.beam.
will.wander.around.the.back.aperture..It.is.clear.why.the.scanners.and.objective.need.to.be.conjugate.but.
perhaps.this.is.not.so.obvious.for.the.AO.element..his.can.clearly.be.recognized.if.one.considers.two.of.
the.simplest.aberrations:.those.of.tip.and.tilt..Clearly,.if.the.AO.element.needs.to.add.a.tip.to.the.beam.to.
compensate.for.a.sample.aberration,.it.will.move.the.beam.over.the.back.aperture.of.the.objective.unless.
the.two.are.conjugate..he.same.thinking.also.applies.to.all.the.complex.aberrations.and.thus.adjusting.
the.optics.to.ensure.all.the.relevant.optical.planes.are.correctly.imaged.onto.one.another.is.crucial.and.
indeed.the.most.diicult.alignment.task.in.an.AO.microscope.system.
A.typical.postscanned.system.is.shown.in.
Figure.11.2
,.illustrating.the.key.elements.of.previously.pub-
lished.systems.(Marsh.et.al..2003).in.a.block.diagram..he.light.from.the.laser.is.directed.straight.into.
the.beam-scanning.head..In.the.diagram,.the.typical.1.mm.laser.beam.does.not.need.to.be.expanded,.
or.contracted,.onto.the.3.mm.scanning.mirrors..he.output.from.the.scan.head.is.then.reimaged.onto.
the.AO.element.using.beam.expansion.if.required..In.the.coniguration.shown,.the.coupling.onto.and.
of.the.AO.element.is.provided.via.polarization.optics..he.incoming.light.passes.through.a.polarizing.
beam.splitter,.through.a.quarter.waveplate,.and.onto.the.mirror..On.relection,.the.beam.passes.through.
the.waveplate.a.second.time.so.that.it.is.now.relected.by.the.beam.splitter.before.being.directed.onto.the.
objective.lens.such.that.the.AO.element.is.conjugate.with.the.back.aperture..In.these.reimaging.optics,.
the.beam.size.can.be.adjusted.to.ensure.optimal.throughput.of.excitation.light.as.described.earlier.
An.alternative.coniguration.is.to.place.the.AO.element.before.the.scanning.element.(Wright.et.al..
2007),. with. the. block. diagram. in.
Figure. 11.3
. showing. the. key. components.. Typically,. the. beam. now.
needs.to.be.expanded.onto.the.AO.element.and.then.reduced.in.size.as.it.is.reimaged.onto.the.scanning.
