Biomedical Engineering Reference
In-Depth Information
lenses.with.the.same.NA.(1.2).and.detectors.with.the.same-size.pinholes.(0.9.Airy.units)..he.transverse/
axial.resolutions.were.improved.from.0.32/1.42.μm.for.the.commercial.system.to.0.23/0.8.μm.for.our.
AO. system.ater.correction.. Although. reducing. the. pinhole. diameter. can. increase. the. resolution,. the.
commercial.system.still.sufers.from.aberration..he.lateral/axial.resolution.for.the.commercial.system.
with.0.5.Airy.units.is.0.29/1.11.μm.
17.4 Using Fluorescent Proteins as Guide Stars
To.make.a.stable.and.fast.wavefront.measurement,.a.direct.wavefront.measurement.method.has.been.
demonstrated,.where.a.luorescent.microsphere.injected.into.the.sample.was.used.as.a.reference.source.
for.a.SHWF.sensor.(Azucena.et.al..2011;.Tao.et.al..2011a,.b)..he.exposure.time.during.wavefront.sensing,.
typically.35.ms,.is.much.shorter.than.the.indirect.wavefront.methods.(Debarre.et.al..2009;.Ji,.Milkie,.&.
Betzig.2010),.which.reduces.the.possibility.of.phototoxicity.and.photobleaching..It.also.enables.higher-
speed. imaging. for. dynamic. live. samples.. However,. this. method. requires. the. injection. of. luorescent.
microspheres.into.the.sample,.which.complicates.the.sample.preparation.procedure..he.microspheres.
are.required.to.cover.the.whole.region.of.interest,.and.the.distance.between.two.microspheres.should.be.
less.than.the.isoplanatic.width.(Azucena.et.al..2010)..As.an.invasive.method,.the.side.efects.of.injection.
to.the.functionality.of.the.biological.tissue.need.to.be.considered.for.live.imaging.
To.overcome.these.disadvantages.and.generalize.the.direct.wavefront.sensing.method,.we.use.luo-
rescent.proteins.as.laser.guide-stars.(Tao.et.al..2011c)..Here.we.use.luorescent.proteins.that.label.par-
ticular.cellular.structures.rather.than.speciic.layers.such.as.the.retina.in.the.eye.(Biss.et.al..2007;.Diaz.
Santana.Haro.&.Dainty.1999)..An.example.is.green.luorescent.protein.(GFP),.from.the.bioluminescent.
jellyish.
Aequorea victoria
.(Lakowicz.2006)..he.luorophore.unit.is.located.within.a.barrel.of.β-sheet.
protein,.which.shields.the.chromophore.from.the.local.environment..Because.of.this.special.structure,.
GFP. has. good. photostability. and. high. quantum. yields. (Lakowicz,. 2006).. his. feature. makes. GFP. a.
good. candidate. as. a. laser. guide-star. in. AO. microscopy.. As. a. noninvasive. luorescent. marker,. GFP.
has.been.extensively.used.in.live.cell.imaging..AO.microscopy.can.be.easily.applied.to.those.studies.
without.special.preparation.of.the.samples..he.possible.damage.to.the.sample.from.the.injection.of.
a.guide-star.reference.bead.can.be.avoided..Here.the.genetic.mutant.of.GFP,.YFP.yellow.luorescent.
protein,.was.used.for.wavefront.measurement.(Patterson.et.al.,.1997).
17.4.1 Method
Figure. 17.22
.
shows. the. system. setup,. which. integrates. an. AO. system. into. a. confocal. microscope.. A.
solid-state.laser.(λ.=.515nm).provides.the.excitation.light.for.both.luorescence.imaging.and.wavefront.
sensing. instead. of. separate. lasers. (515. and. 633. nm). for. the. system,. shown. above. in.
Figure. 17.16
.
. he.
light.is.fed.into.an.objective.lens.(×60.water.objective,.NA.1.2,.Olympus).and.scanned.on.the.sample.in.
a.raster.pattern.with.two.galvo.scanners.(6215H,.Cambridge.Technology)..he.emission.light.from.the.
luorescent.protein.is.divided.by.a.BS.with.a.50/50.splitting.ratio..Half.of.the.light.was.collected.by.the.
PMT.(H422-50,.Hamamatsu),.which.generates.the.confocal.image..he.other.half.of.the.light.is.used.for.
wavefront.sensing..We.are.investigating.the.use.of.a.lip-mirror.to.guide.100%.of.the.luorescent.light.
to.the.SHWF.sensor.for.wavefront.sensing.or.to.the.PMT.for.imaging.to.avoid.splitting.the.light.50/50.
with.the.BS..Four.relay.lens.groups.make.the.exit.pupil.of.the.objective.lens.conjugate.with.the.SHWF.
sensor,.DM,.and.scanners..To.minimize.the.amount.of.out-of-focus.light.that.enters.the.SHWF.sensor,.
irises.I1.and.I2.were.placed.in.the.light.path..hese.irises.also.block.stray.light.from.the.DM,.scanner,.
and.lenses..he.position.of.the.spots.in.the.image.from.the.wavefront.sensor.was.detected.using.a.cross-
correlation. centroiding. algorithm. (homas. et. al.,. 2006).. hen. an. FFT. reconstruction. algorithm. was.
implemented. to. obtain. the. wavefront. (Poyneer,. 2003).. To. make. an. accurate. wavefront. measurement.
using. a. SHWF. sensor,. the. diameter. of. the. guide. star. should. be. smaller. than. the. difraction. limit. of.
the.wavefront.sensor,.which.is.deined.in.Equation.17.1..In.our.current.setup,.
d
difraction_limit
.=.5.36.μm..
