Biomedical Engineering Reference
In-Depth Information
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hus,.any.increase.in.the.focal.volume,.from.whatever.source,.will.signiicantly.decrease.the.expected.
signal.to.the.sixth.power.of.the.radius,.assuming.a.spherical.excitation.volume..he.squared.terms.come.
from.two-photon.luorescence.imaging;.in.the.case.of.three-photon.or.high-order.excitation.methods,.
this.becomes.cubed.or.higher..It.is.thus.clear.that.minimizing.the.excitation.volume.plays.a.signiicant.
role.in.maximizing.the.signal.to.noise.in.nonlinear.microscopy.and.determining.the.maximum.possible.
resolution.
11.1.2 Sources of Aberration
It.is.worth.briely.considering.the.root.cause.of.increases.in.the.excitation.volume..In.general,.there.are.
two.main.sources.of.aberrations.in.nonlinear.microscopy.and.the.level.of.contribution.is.not.exactly.the.
same.as.in.the.case.of.linear-excitation-based.systems..he.irst.and.most.obvious.source.of.aberrations.
is.sample-related.because.of.the.local.changes.in.refractive.index.causing.diferent.parts.of.the.excitation.
wavefront. to. be. delayed. relative. to. other. areas,. leading. to. a. signiicantly. enlarged. or. nondifraction-
limited.spot.at.the.focal.point.of.the.system..hese.aberrations.become.larger.with.depth.and.are.also.
very.sample-dependent..he.use.of.longer.wavelength.excitation.sources.required.for.nonlinear.imaging.
methods.tends.to.mean.this.efect.is.slightly.lower.than.that.for.single-photon.excitation,.but.the.difer-
ence.is.small..he.main.advantage.of.longer.wavelength.in.this.context.is.that.scattering,.which.is.very.
wavelength-dependent,.is.reduced.in.the.sample..Full.details.on.these.efects.can.be.found.in.Chapter.5.
of.this.book.or.summarized.in.a.recent.review.(Girkin.et.al..2009).
he.other.signiicant.sources.of.aberrations.in.a.nonlinear.microscope.are.those.present.in.the.system..
Although.all.beam-scanned.microscopes.are.optimized.for.performance,.some.compromise.has.to.be.
made.in.the.design.process,.perhaps.trading.of.resolution.for.improved.light.throughput.or.balancing.
out.chromatic.aberration.efects.because.of.the.large.diference.in.excitation.and.emission.light.in.such.
microscopes..In.addition,.over.time.even.in.the.best-maintained.microscope.components.drit.slightly,.
causing.aberrations.that.may.not.be.obvious.to.facility-based.users..he.use.of.diferent.objectives.can.
also.increase.the.source.of.such.aberrations.and.AO.provides.one.method.to.ensure.that.systems.are.kept.
optimized.even.before.sample.aberrations.add.further.complications.in.the.imaging.path.
here.is.one.further.source.of.aberration.that.is.present.in.nonlinear.imaging.systems,.which.is.not.
normally. seen. in. more-conventional. single-photon. excitation. microscopes:. beam. distortions. present.
in. the. laser. source. itself.. In. conventional. microscopes,. the. excitation. light. is. delivered. either. directly.
from.the.laser.or,.more.commonly.now,.via.a.single-mode.optical.iber..his.means.that.the.light.enter-
ing.the.scanning.system.has.an.intensity.proile.and.wavefront.determined.by.the.optical.iber,.which.
will. in. general. be. round,. nonastigmatic,. and. typically. have. a. Gaussian. intensity. proile.. he. spectral.
bandwidth. of. the. laser. source. will. also. be. small,. and. even. when. several. laser. sources. with. diferent.
wavelengths.are.delivered.simultaneously,.the.wavelength.range.is.frequently.less.than.100.nm..But.this.
is.not.the.case.with.nonlinear.systems.
To.achieve.femtosecond.laser.pulses,.the.laser.cavity.of.Ti:Sapphire.lasers.contains.a.number.of.extra.
optical.elements.(Girkin.2008)..hese.will.not.be.described.in.detail.here.but.crucially.they.add.distor-
tion.to.the.output.beam.of.the.laser,.frequently.making.it.astigmatic..When.this.beam.is.then.expanded.
and. manipulated. through. the. beam-scanning. optics,. the. optical. train. can. increase. these. aberrations.
leading. to. large. focal. volumes.. In. addition,. the. broad. spectrum. of. the. laser. pulse. (typically. around.
50 nm.for.a.center.wavelength.of.800.nm),.even.before.the.laser.tuning.is.considered.(680-1060.nm.in.a.
typical.modern.Ti:Sapphire.laser),.adds.to.this.complication..hus,.the.light.source.is.adding.to.the.aber-
rations.that.need.to.be.corrected..Recently,.the.use.of.AO.to.remove.these.efects.has.been.demonstrated.
in.a.two-photon.microscope.used.for.imaging.ocular.tissue.(Gualda.et.al..2010).
