Biomedical Engineering Reference
In-Depth Information
11.1.1 Basics of nonlinear imaging
Before.considering.the.exact.role.that.AO.now.plays.in.nonlinear.optical.microscopy,.it.is.worth.consid-
ering.the.basic.physics.behind.the.nonlinear.imaging.methodology.as.this.really.sets.the.requirements.
for. the. adaptive. system.. he. original. underpinning. theoretical. physics. behind. multiphoton. luores-
cence.microscopy.was.developed.nearly.80.years.ago.by.Prof..Göppert-Mayer.(1931),.as.she.worked.in.
the. then. rapidly. emerging. new. ield. of. quantum. mechanics.. he. concept. that. two. photons. could. be.
absorbed.simultaneously.to.cause.an.electron.transition.within.an.atom.or.a.molecule.was.at.the.time.
very. much. a. curiosity. of. theoretical. physics.. Practical. use. of. this. predication. was. not. possible. until.
the. laser. was. invented. in. 1960. leading. to. the. development. of. nonlinear. optics,. a. technique. also. now.
used.in.microscopy.and.multiphoton.absorption.for.high-resolution.laser.spectroscopy..he.concept.of.
nonlinear.microscopy,.where.the.returned.signal.is.not.linearly.dependent.on.the.excitation.intensity,.
and.more.than.one.photon.is.required.for.the.excitation.process,.was.demonstrated.in.the.1970s,.but.
the.main.complication.was.the.laser.power.required.to.generate.suicient.signal.without.destroying.the.
sample..Q-switched.lasers.had.the.energy.required.but.the.length.of.pulse.tended.to.lead.to.smoke.and.
dust.rather.than.an.intact.sample.ater.imaging.
As.laser.physicists.developed.ever.shorter.pulse.lasers,.the.possibility.of.a.source.with.very.high.peak.
power.but.low.average.power.became.a.reality,.leading.to.the.irst.article.on.two-photon.luorescence.
microscopy.by.Denk.et.al..(1990)..he.use.of.femtosecond.pulses,.albeit.from.a.colliding.pulse.dye.laser,.
meant.that.the.average.power.on.the.sample.was.low,.leading.to.minimal.thermal.damage;.the.energy.
in.each.pulse.was.also.low.minimizing.explosive.damage,.but.the.photon.density.was.high.leading.to.
suicient.two-photon.excitation.events..As.this.nonlinear.absorption.happens.only.at.the.focus.of.the.
excitation.beam,.the.method.provides.inherent.optical.sectioning,.enabling.three-dimensional.images.
to.be.built.up,.although.clearly.the.size.of.that.focal.volume.is.crucial.to.the.resolution.and.number.of.
excitation.events.for.a.given.laser.power.
Such. three-dimensional. optical. images. were,. however,. already. available. through. confocal. micros-
copy..he.crucial.advantage.of.the.nonlinear.approach.was.the.use.of.longer.wavelength.light.that.could.
penetrate.further.into.samples.because.of.the.reduced.scattering.and.also,.in.general,.was.not.directly.
absorbed.by.endogenous.luorophores..he.longer.wavelength.light.also.signiicantly.reduced.the.risk.of.
phototoxic.damage.that.was.always.present.if.ultraviolet.light.was.used..At.this.stage.in.the.development.
of.nonlinear.microscopy,.the.major.downside.to.the.method.was.the.requirement.for.a.laser.producing.
ultrashort.pulses.in.the.near.infrared..he.Ti:Sapphire.laser.provided.a.solution.to.this.problem.and.the.
source.issue.was.further.helped.by.the.signiicant.improvements.that.were.being.made.at.the.time.in.
laser.diodes.for.pumping.Nd-based.laser.sources,.which.when.frequency.doubled.were.rapidly.replacing.
the.argon.ion.laser.pump.source.(Girkin.and.McConnell.2005).
With. such. sources. becoming. routinely. available,. the. concept. of. using. nonlinear. optical. methods. for.
imaging,. with. their. inherent. sectioning,. grew. rapidly. with. the. movement. toward. imaging. without. add-
ing.exogenous.luorophores.and.second.harmonic,.third.harmonic.imaging.of.live.biological.tissue.arrived.
along. with. coherent. anti-Stokes. Raman. scattering. (CARS),. in. which. direct. molecular. vibration. could.
be.excited.within.molecules.already.present.in.the.tissue.(Zumbusch.et.al..1999)..One.other.crucial.point.
that.should.be.noted.is.that.in.parallel.with.the.technological.developments.came.molecular.and.genetic.
advances.in.biology,.most.speciically.the.development.of.luorescent.proteins.that.could.be.expressed.very.
speciically.within.living.cells.with.minimal.perturbation.to.the.biology.(except.for.the.presence.of.a.large.
nonendogenous.protein),.producing.a.desire.for.the.life.scientists.to.image.long.term,.in.vivo,.and.at.depth.
Now.I.will.consider.the.basics.of.all.nonlinear.imaging..For.a.given.number.of.molecules.capable.of.
excitation. (
N
),. the. number. actually. excited. is. given. by.
N
. times. the. probability. of. an. excitation. event.
taking.place,.
P
excite
..his.term.depends.on.several.factors:.the.instantaneous.peak.power.
P
peak
,.which.is.a.
function.of.the.average.power.
P
cw
;.the.pulse.length.τ;.repetition.rate.
f
;.the.multiphoton.absorption.cross.
section.at.the.given.wavelength.σ;.and.from.our.perspective.inversely.with.the.excitation.volume.
V
.
2
..In.
total,.this.gives
