Biomedical Engineering Reference
In-Depth Information
Point-scanning
Wide-field
Objective
Objective
Aberrated
wavefront
(a)
(b)
FIGuRE 13.1 Schematics. depicting. the. efects. of. optical. inhomogeneities. on. the. excitation. light. in. a. point-.
scanning.microscope.( let ).and.the.emission.light.in.a.wide-ield.microscope.( right )..Arrowed.lines.describe.light.
ray.propagation.direction.and.the.curves.intersecting.the.arrowed.lines.denote.wavefronts.
sample. heterogeneities. that. give. rise. to. image. contrast. also. cause. spatial. variations. in. their. opti-
cal. properties.. For. example,. biological. samples. are. comprised. of. structures. (i.e.,. proteins,. nuclear.
acids,.lipids).with.diferent.refractive.indices.[2]..It.is.all.but.impossible.to.design.a.microscope.with.
an. immersion. medium. matching. these. varying. refractive. indices. simultaneously.. herefore,. these.
samples. induce. optical. aberrations. on. both. the. incoming. and. outgoing. waves.. For. point-scanning.
microscopes,.such.as.a.two-photon.luorescence.microscope,.the.aberrations.of.the.excitation.light.
result. in. an. enlarged. focal. spot. within. the. sample. and. a. concomitant. deterioration. of. signal. and.
resolution..For.a.wide-ield.microscope,.the.wavefront.from.the.luorescence.emission.at.each.point.
in.the.sample.is.similarly.distorted,.preventing.a.difraction-limited.image.from.being.formed.at.the.
image.detector.(Figure.13.1).
In. either. case,. difraction-limited. performance. may. be. restored. using. active. optical. components,.
such.as.a.spatial.light.modulator.(SLM).or.a.deformable.mirror,.to.modify.the.excitation.or.emission.
wavefront. in. such. a. way. as. to. cancel. out. the. sample-induced. aberrations. [3].. Such. approaches,. col-
lectively.named.adaptive.optics.(AO),.were.initially.developed.in.astronomy.to.combat.the.aberrations.
caused. by. the. earth's. atmosphere. on. the. wavefronts. arriving. from. extraterrestrial. sources. [4-6].. A.
wavefront.sensor,.usually.made.of.a.2D.array.of.lenses.focused.on.the.diferent.subregions.of.a.camera.
and.incorporated.into.a.feedback.loop.with.a.deformable.or.segmented.mirror,.measures.the.wavefront.
directly.and.dictates.the.surface.form.of.the.mirror.
In.microscopy,.such.direct.wavefront.sensing.is.not.generally.applicable..For.in.vivo.imaging.with.a.
point-scanning.microscope,.it.is.usually.not.possible.to.use.a.wavefront.sensor.to.measure.the.excita-
tion.light.wavefront.directly.ater.the.aberrating.sample..For.samples.with.high.optical.transparency,.
such. as. the. human. eyes,. direct. wavefront. sensing. schemes. using. relected. light. have. been. successful.
[7, 8,.Section.IV.of.this.book]..However,.the.strong.scattering.of.most.biological.samples.prevents.direct.
wavefront. sensing. from. being. widely. applicable.. Indirect. wavefront-sensing. schemes. are. needed. as. a.
result..In.this.chapter,.we.describe.two.pupil-segmentation-based.AO.methods,.where.we.measure.the.
wavefront.aberration.segment.by.segment..his.zonal.approaches.they.difer.from.the.zonal.wavefront.
sensing.method.in.astronomy.in.that.they.are.indirect,.image/signal-based.methods,.which.makes.them.
applicable.to.strongly.scattering.samples,.such.as.mouse-brain.tissues.
13.2 Adaptive optical two-Photon Microscopy
Using Pupil Segmentation
Our. approach. is. based. on. a. simple. physical. picture. of. focal. formation—a. focus. arises. from. a. spheri-
cal. wavefront,. wherein. all. light. rays. are. bent. to. converge. at. a. common. point. with. a. common. phase,.
resulting.in.maximally.constructive.interference,.and.thus.a.difraction-limited.focus.[9].( Figure.13.2a )..
Within.an.optically.inhomogeneous.sample,.however,.rays.traversing.regions.with.diferent.refractive.
 
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