Biomedical Engineering Reference
In-Depth Information
previously.mentioned.examples.in.Chapter.7,.an.AO.solution.has.been.implemented..AO.has.been.suc-
cessfully.implemented.in.both.confocal.and.nonlinear.microscopy.techniques,.such.as.two-photon.excita-
tion.microscopy,.harmonic.generation.microscopy,.and.coherent.anti-Stokes.Raman.scattering.(CARS).
microscopy.[5].
In.an.AO.system,.the.wavefront.distortions.can.be.compensated.with.an.active.element,.and.the.resul-
tant.image.resolution.and.signal.level.can.be.maintained.to.greater.imaging.depths..However,.the.issue.
is. not. just. to. correct. the. wavefront. distortions. (aberrations). but. also. to. characterize. and. measure. the.
.distortions,. without. which. the. correction. of. a. single. scanned. position. of. the. focused. beam. can. take.
several. minutes.. he. detection. of. aberrations. in. AO. microscopy. can. be. categorized. into. two. main.
approaches—wavefront. sensorless. and. wavefront. sensors.. Of. the. two,. the. former. does. not. employ. a.
wavefront.sensor.(e.g.,.SHWS),.but.instead.relies.on.a.chosen.imaging.metric,.such.as.the.excited.luo-
rescence.signal.strength.or.image.sharpness..With.the.latter,.aberrations.are.detected.from.the.relected/
backscattered.excitation.light.using.a.wavefront.sensor..In.the.latter.method,.care.must.be.taken.because.
the.excitation.light.passes.through.the.aberrating.sample.twice.and.it.is.diicult.to.distinguish.between.
odd.and.even.aberrations.ater.relection..In.the.following.sections,.we.describe.the.main.diferences.and.
various.implementations.of.the.two.methods.of.detecting.wavefront.aberrations.in.microscopy.
16.2 Methods and experimental Setup
A.homebuilt.two-photon.microscope.was.set.up.following.the.principles.laid.out.[6]..he.implementa-
tion. of. AO. was. through. a. large. dynamic. range. deformable. mirror. and. a. relected. light. path. with. a.
confocal.pinhole.before.the.SHWS.
16.2.1 Large Dynamic range Deformable Mirror
he. deformable. mirror. (Mirao52d;. Imagine. Optic,. Orsay,. France). includes. a. silver-coated. relective.
membrane.coupled.with.52.miniature.voice-coil-type.actuators..he.diameter.of.its.pupil.is.15.mm,.and.
it. has. a. 200. Hz. bandwidth.. he. maximum. wavefront. amplitude. that. can. be. generated. is. 50.μm. with.
1-V.voltage.range..An.important.feature.of.this.deformable.mirror.is.its.large.dynamic.range,.and.it.can.
generate.Zernike.modes.up.to.the.fourth.order.and.partially.up.to.the.sixth.order.
16.2.2 Detection of Wavefront Distortion with a
Shack-Hartmann Wavefront Sensor
he.SHWS.(HASO32;.Imagine.Optic).consists.of.a.lenslet.array.and.a.charge-coupled.device.(CCD).as.
the.sensor..It.has.a.32.×.32.lens.array.within.a.3.6.×.3.6.mm
2
.rectangular.aperture.and.a.bandwidth.of.20.
Hz..Each.lenslet.produces.a.focal.spot.that.is.detected.over.a.region.on.the.CCD-that.is,.a.group.of.pixels.
(sub-detection.area).corresponding.to.the.aperture.of.the.lenslet..If.the.incident.wavefront.is.plane,.the.
foci.are.all.centered.on.the.appropriate.pixels..If.the.wavefront.is.aberrated,.the.foci.shit.across.the.sub-
detection.area.according.to.the.type.of.aberration..he.slope.of.the.wavefront.is.given.by.the.ratio.of.the.
shit.over.the.focal.length.of.the.lenslet,.and.the.whole.wavefront.can.be.reconstructed..he.advantage.
of.the.Shack-Hartmann.sensor.is.its.ability.to.rapidly.quantify.wavefront.aberration.with.a.single.image.
16.2.3 Application of confocal Detection to Select
relected Light Signals from the Focal Plane
In. the. case. of. luorescence. microscopy,. only. the. signal. originating. from. the. vicinity. of. the. focus. is.
relevant.for.the.purpose.of.aberration.detection.and.correction.[7-9]..In.a.real.biological.sample,.scat-
tering.and.other.inhomegeneities.of.the.refractive.index.mean.that.sample-induced.aberrations.are.
