Biomedical Engineering Reference
In-Depth Information
1998)..AO.might.have.been.conceived.for.the.purpose.of.improving.astronomical.imaging,.but.other.
scientists. soon. realized. the. importance. of. this. technology. in. other. areas. of. research.. In. particular,.
vision. science. was. one. of. those. ields. where. AO. has. enlightened. curious. researchers.. he. irst. major.
obstacle.in.adapting.AO.to.vision.science.was.to.ind.a.reasonable.reference.source.for.measuring.the.
wavefront..he.irst.Shack-Hartmann.wavefront.(SHWF).sensor.measurements.for.vision.science.were.
realized. by. Junzhong. Liang. by. imaging. a. laser. spot. onto. the. retina. (Liang. et. al.. 1994).. A. few. years.
later,.Liang,.Williams,.and.Miller.(1997).inally.constructed.the.irst.closed-loop.AO.system.for.vision.
science.
he. idea. of. using. AO. for. microscopes. is. relatively. new. and. a. lot. of. work. is. still. needed.. Most. AO.
microscopy.systems.have.so.far.not.directly.measured.the.wavefront.because.of.the.complexity.of.add-
ing.a.wavefront.sensor.in.an.optical.system.and.the.lack.of.a.natural.point-source.reference.such.as.the.
“guide.star”.used.in.astronomy..Instead,.most.AO.microscopy.systems.have.corrected.the.wavefront.by.
optimizing. a. signal. received. at. a. photodetector. (Booth. 2007).. Debarre. et. al.. (2008). have. successfully.
incorporated.an.AO.system.into.a.structured.illumination.microscope..In.his.research,.Debarre.used.a.
wavefront.sensorless.AO.technique.in.which.each.mode.is.corrected.independently.through.the.sequen-
tial.optimization.of.an.image-quality.metric..Scientists.and.engineers.have.also.been.investigating.ways.
to.implement.AO.in.two-photon.microscopes.(Marsh,.Burns,.&.Girkin.2003;.Rueckel,.Mack-Bucher,.&.
Denk.2006)..Rueckel.et.al..used.a.coherence.gate.to.selectively.pass.only.the.light.backscattered.near.the.
focus.to.directly.measure.the.wavefront.using.an.interferometer.
Although.there.are.a.lot.of.important.researches.being.done.in.AO.microscopy,.many.of.the.AO.sys-
tems.are.speciic.to.each.microscope,.and.a.universal.method.for.measuring.the.wavefront.(or.the.results.
of.the.correction.algorithm).is.not.currently.available..Booth.(2007).described.some.of.the.diiculties.
associated.with.the.utilization.of.a.SHWF.sensor.in.AO.microscopy..Most.of.these.diiculties.can.be.
overcome. if. a. suitable. luorescent. point-source. could. be. found.. Beverage. and. others. found. a. suitable.
method.for.measuring.the.wavefront.of.a.microscope.objective.lens.by.using.luorescent.microspheres.
as.reference.sources.(Beverage,.Shack,.&.Descour.2002)..In.his.research,.Beverage.established.that.bigger.
beads.(larger.than.difraction.limit).could.be.used,.allowing.for.more.light.to.measure.the.wavefront..
he.size.of.the.beads,.
d
bead
,.should.be.smaller.than.the.difraction.limit.of.the.wavefront.sensor.when.
imaged.through.the.microscope.objective:
λ
D
d
o
.
d
=
2 44
2
.
=
d
N
D d
.
.(17.1)
bead
DLO
⋅
/
NA
ob
LA
where.λ.is.the.wavelength.at.which.the.beads.are.emitting,.NA
ob
.is.the.numerical.aperture.(NA).of.the.
objective,.
D
o
.is.the.limiting.aperture.of.the.objective,.and.
d
LA
.is.the.lenslet.array.pitch..his.could.also.be.
represented.as.the.difraction.limit.of.the.objective.(
d
DLO
).times.the.number.of.subapertures.across.the.
limiting.pupil..Using.this.technique,.we.can.measure.the.aberration.introduced.by.a.biological.sample.
by.injecting.a.luorescent.bead.into.the.sample..To.reduce.the.efect.of.the.scattered.light,.a.ield.stop.
can.be.used..he.ield.stop.can.be.placed.at.the.image.focal.plane.just.before.the.wavefront.sensor..he.
system.must.be.designed.so.that.only.the.wavefront.sensor.sees.the.ield.stop..he.ield.stop.will.act.as.a.
spatial.ilter,.so.that.the.wavefront.sensor.will.not.see.spatial.frequencies.above.the.size.of.the.ield.stop..
To.reduce.aliasing.at.the.SHWF.sensor,.the.size.of.the.ield.stop.cannot.be.smaller.than.2
d
LA
.as.projected.
onto.the.image.plane.(Poyneer,.Gavel,.&.Brase.2002).
17.1.1 Fluorescent Beads
Fluorescent.microspheres,.with.a.large.variety.of.colors,.are.typically.used.in.biology.to.study..diferent.
biological.characteristics.(Invitrogen.Corporation.2010;.Rothwell.&.Sullivan.2000;.Guldband.et.al..2010;.
DeMarais,.Oldis,.&.Quatrro,.2005;.Rowning.et.al..1997;.Kalpin,.Daily,.&.Sullivan.1994)..he.microspheres.
are. made. of. polymers. and. are. impregnated. with. diferent. luorescent. dyes.. he. microspheres. can. be.
