Biomedical Engineering Reference
In-Depth Information
wavefront.sensor..he.SHWF.sensor.is.composed.of.a.44.×.44.element.lenslet.array.with.a.lenslet.diam-
eter.of.400.μm.and.focal.length.of.24.mm.(AOA.Inc.,.Cambridge,.MA).and.an.electron.multiplying.CCD.
(EMCCD).camera.(Cascade.II,.Photometrics)..he.dichroic.BS.DB2.separates.the.light.from.the.crimson.
reference.beacon.and.the.YFP-labeled.sample..he.dichroic.BSs.DB1.and.DB3.are.used.to.separate.the.
excitation.light.and.its.associated.back.relection..he.luorescent.light.emitted.by.the.YFP.is.iltered.by.
F3.and.detected.by.the.photomultiplier.tube.(PMT)..he.wavefront.aberration.is.corrected.by.the.DM.
Two.laser.channels.are.included.in.the.system:.a.solid-state.laser.(λ.=.515.nm).and.a.helium-neon.laser.(λ.=.
633.nm).used.for.confocal.luorescence.imaging.and.wavefront.sensing,.respectively..he.two.channels.share.
the.same.light.path.through.the.DM,.relay.lenses,.scanner,.and.scanning.lens.and.feed.into.an.Olympus.IX71.
inverted.microscope.(Olympus.Microscope,.Center.Valley,.PA).through.its.side.optical.port..he.focused.
beam.is.scanned.on.the.sample.in.a.raster.pattern.with.a.resonant.scanner.(SC-30,.16.kHz,.5°,.Electro-Optics.
Products.Corp.).and.a.vertical.scanner.(GVS001,.horlabs)..he.control.signals.for.the.scanners.are.gener-
ated.by.a.data-acquisition.board.(PCIe-6363,.National.Instruments.Corporation)..he.emission.light.from.
both. the. crimson. microsphere. guide-star. and. the. YFP-labeled. sample. are. collected. by. an. objective. lens.
(Olympus.60×,.NA1.2.water.immersion).and.separated.by.a.dichroic.mirror.(DB2)..he.light.from.the.sam-
ple.was.focused.onto.a.pinhole.by.an.achromatic.lens.L10..A.GaAs.PMT.(H422-50,.Hamamatsu).was.used.
as.a.photon.detector..he.signal.from.the.PMT.is.then.fed.to.a.frame-grabber.board.(Helios.eA/XA,.Matrox.
Imaging)..With.the.vertical.and.horizontal.synchronized.signal.from.the.DAQ,.the.frame-grabber.board.
generates.a.raw.image.of.512.×.512.pixels.at.30.frames/second.for.live.imaging..When.the.signal.levels.are.
low,.more.frames.can.be.averaged.to.increase.the.signal-to-noise.ratio.(SNR)..We.have.also.used.two.galvo.
scanners.(6215H,.Cambridge.Technologies).at.lower.frame.rates.(3.Hz).to.increase.the.SNR..he.experimen-
tal.system.was.setup.on.an.optical.table.with.vibration.isolation.as.shown.in.Figure.17.17..he.beam.from.the.
scanning.system.was.fed.into.the.Olympus.IX71.inverted.microscope.by.a.periscope.
17.3.2 results for Fixed Mouse-Brain tissue
To.investigate.the.feasibility.of.the.proposed.system,.a.ixed.brain.slice.from.a.YFP-H.line.transgenic.
mouse. was. prepared.. YFP. is. labeled. on. the. cell. body. and. protrusions. of. the. neurons.. One. micron.
Olympus X61 Frame
PMT
Science
camera
HeNe laser
Solid state laser
DM
SH Wavefront
sensor
X Scanner
Y Scanner
FIGuRE 17.17
Experimental.setup.of.an.adaptive.optics.confocal.microscope..he.system.was.setup.on.an.optical.
table.with.vibration.isolation..he.optical.path.for.the.crimson.reference.beacon.(guide.star).is.shown.as.a.white.
solid.line..A.HeNe.laser.is.used.for.excitation.of.the.guide.star.and.its.emission.wavefront.is.measured.with.a.Shack-
Hartman.wavefront.sensor..he.optical.path.from.the.microscope.to.the.photomultiplier.tube.(PMT).for.detecting.
the.sample.luorescent.emission.(YFP).is.shown.as.a.dashed.line..he.excitation.path.for.the.sample.(515.nm.solid-
state.laser).is.shown.as.a.grey.solid.line.
