Biomedical Engineering Reference
In-Depth Information
13
Pupil-Segmentation-Based
Adaptive Optics for
Microscopy
13.1.
Introduction.......................................................................................231
13.2.
Adaptive.Optical.Two-Photon.Microscopy.Using.Pupil.
Segmentation......................................................................................232
13.3.
Experimental.Setup...........................................................................236
13.4.
Results.................................................................................................237
Examples
13.5.
Discussion...........................................................................................243
Scattering.Control
Na Ji
Howard Hughes
Medical Institute
Eric Betzig
Howard Hughes
Medical Institute
13.6.
Summary............................................................................................249
13.1 introduction
Optical. microscopy. has. long. been. used. to. study. objects. too. minuscule. for. the. naked. human. eye. [1]..
hese.minute.bodies.almost.always.have.spatially.heterogeneous.optical.properties..For.example,.their.
compositions.difer.in.relectivity,.absorption,.scattering,.or.luorescence..hese.inhomogeneities.pro-
vide.contrast.in.micrographs.and.are.oten.themselves.the.center.of.investigation..Because.of.the.wave.
nature.of.light,.however,.the.resolving.power.of.conventional.optical.microscopy.is.limited.by.difrac-
tion..Even.in.a.microscope.devoid.of.defects,.a.point.source.appears.as.a.blob.on.its.image.plane.and.
the.illumination.light.can.be.focused.only.into.a.volume.rather.than.a.single.point,.with.the.extent.of.
its.positional.uncertainty.dictated.by.the.point.spread.function.(PSF).of.the.imaging.system..For.a.high.
numerical.aperture.(NA).microscope.operating.in.the.visible.range,.the.difraction-limited.resolution.is.
on.the.order.of.hundreds.of.nanometers.
In.practice,.however,.difraction-limited.resolution.is.oten.not.achieved..his.is.because.micro-
scopes.are.designed.to.operate.at.the.difraction.limit.only.in.samples.with.speciic.optical.properties..
For.example,.an.oil-immersion.objective.forms.perfect.images.only.for.objects.on.the.distant.surface.
of. a. coverglass. of. a. certain. thickness. and. with. its. other. surface. immersed. in. oil;. a. water-dipping.
objective. can. focus. light. only. to. a. difraction-limited. volume. in. water.. Deviations. from. such. rig-
orous. requirements. are. common—coverglasses. have. variations. in. their. thickness,. and. the. immer-
sion.media.may.be.at.a.temperature.that.causes.its.refractive.index.to.difer.from.the.speciication..
More.inevitably,.the.structures.we.would.like.to.visualize.degrade.image.quality.by.themselves—the.
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