Biomedical Engineering Reference
In-Depth Information
PMT
Pinhole
Solid-state
laser
(515 nm)
F2
Objectives
F3
SHWS
DB
BS
I1
F1
X Scanner
BB
DM
I2
Y Scanner
FIGuRE 17.22
AO.confocal.microscope.setup.for.using.luorescent.protein.guide-stars..A.solid-state.laser.(515.
nm).is.used.as.the.excitation.light.source.for.YFP..A.dichroic.beam.splitter.(DB).separates.the.emission.and.excita-
tion.light..Light.emitted.from.a.YFP-labeled.cellular.structure.is.collected.by.an.objective.lens.and.split.for.wavefront.
measurement.and.confocal.imaging.by.a.50/50.BS..he.wavefront.error.is.measured.by.a.SHWS.and.then.corrected.
by.a.DM..Defocused.and.stray.light.is.blocked.by.irises.I1.and.I2..F2.is.an.excitation.ilter..F3.and.F1.are.emission.
ilters..BB.is.a.beam.blocker..A.PMT.located.behind.a.pinhole.collects.a.confocal.image.of.the.sample's.luorescence.
Because.the.luorescent.light.from.a.given.point.is.proportional.to.the.light.intensity.illuminating.that.
point,.the.size.of.the.guide.star.is.determined.by.the.illumination.PSF..he.PSF.is.calculated.from.the.
wavefront.measurement.using.the.method.described.in.Beverage,.Shack,.and.Descour.(2002)..For.GFP.
at.a.depth.of.70.μm,.the.diameter.of.the.PSF.with.80%.of.the.encircled.energy.is.1.4.μm,.which.is.small.
enough.to.be.used.as.a.guide.star.
To. investigate. the. feasibility. of. the. proposed. system,. a. ixed. brain-tissue. slice. from. a. YFP-H. line.
transgenic.mouse.was.prepared..Sample.brain.coronal.sections.(100.μm).were.cut.with.a.microtome..he.
tissues.were.mounted.on.the.slide.with.antifade.reagents.(Invitrogen)..he.spherical.aberration.induced.
by.the.cover.plate.was.compensated.by.adjustment.of.a.correction.collar.on.the.objective.lens..he.system.
aberration.was.further.corrected.by.the.DM.by.measuring.the.wavefront.aberration.from.the.top.surface.
of.the.tissue..During.the.experiment,.the.confocal.images.were.initially.captured.without.wavefront.cor-
rection..he.approximate.location.of.the.GFP.was.identiied.from.this.initial.image..hen.the.scanning.
mirror.was.used.to.steer.the.laser.beam.to.the.region.of.the.GFP,.and.a.wavefront.measurement.was.per-
formed..he.DM.corrected.the.aberration.in.a.closed.loop.using.the.direct-slope.algorithm.(Porter.et.al..
2006)..hen.the.corrected.confocal.image.of.the.isoplanatic.region.(Azucena.et.al..2010).around.the.GFP.
guide.star.is.captured.with.the.optimal.shape.of.DM.as.determined.from.the.wavefront.measurement.
During.the.wavefront.measurement,.the.laser.is.focused.on.a.stationary.point,.which.may.cause.pho-
tobleaching.of.GFP..By.decreasing.the.laser.power.and.increasing.the.exposure.time.of.the.SHWF.sensor,.
suicient.signal.can.be.captured.by.the.SHWF.sensor.during.the.wavefront.measurement.before.photo-
bleaching..In.this.system,.the.laser.power.is.controlled.by.a.sotware.and.is.turned.down.to.23 nW.at.the.
back.aperture.of.the.objective.lens.during.wavefront.sensing.and.turned.up.to.20.μW.during.confocal.
scanning..he.exposure.time.of.the.SHWF.sensor.could.be.decreased.before.photobleaching.by.increas-
ing.the.laser.power.and.using.a.high-speed.shutter.to.limit.the.exposure.time.of.the.laser.on.the.sample,.
which.will.be.considered.in.the.future..GFP.on.the.dendrite.and.cell.body.of.the.neuron.were.tested.as.
laser.guide-stars.at.depths.of.25.and.70.μm,.respectively,.as.shown.in.
Figure.17.23a
.
and
.
b.
.he.crossed.
lines.show.the.location.of.the.laser.focus..he.exposure.time.of.the.wavefront.measurement.for.the.den-
drite.and.cell.body.is.500.and.30.ms,.respectively..Because.the.dendrites.have.less.GFP.than.the.cell.bod-
ies,.longer.exposure.times.are.required.to.collect.enough.signal.for.accurate.wavefront.measurement..
