Biological Imaging and Adaptive Optics in Microscopy - Adaptive Optics for Biological Imaging - page 299

16
Biological Imaging
and Adaptive Optics
in Microscopy

16.1. Introduction.......................................................................................299

16.2. Methods.and.Experimental.Setup................................................. 300

Large.Dynamic.Range.Deformable.Mirror. •. Detection.of.Wavefront.
Distortion.with.a.Shack-Hartmann.Wavefront.Sensor. •. Application.
of.Confocal.Detection.to.Select.Relected.Light.Signals.from.the.Focal.
Plane. •. Overall.Instrument.Coniguration

Elijah Y. S. Yew
Singapore-MIT Alliance
for Research and
Technology (SMART)

Jae Won Cha
Massachusetts Institute
of Technology

16.3. Experiment.and.Results...................................................................303

Signal.Loss.Due.to.Aberrations.as.a.Function.of.Imaging .
Depth. •. Point.Spread.Function.Degradation.Due.to.Aberrations.as.a.
Function.of.Imaging.Depth. •. Mouse-Tongue-Muscle.Imaging.Using.
Adaptive.Optics-Compensated.Two-Photon.Microscopy. •. Mouse-
Heart-Muscle.Imaging.Using.Adaptive.Optics-Compensated.Two-
Photon.Microscopy. •. Neuronal.Imaging.in.Mouse-Brain.Slices.Using.
Adaptive.Optics-Compensated.Two-Photon.Microscopy

Jerome Ballesta
Imagine Optic

Peter T. C. So
Massachusetts Institute
of Technology 16.4. Conclusions........................................................................................ 311

16.1 introduction

In.general,.adaptive.optics.(AO).for.microscopy.is.no.diferent.from.its.other.applications.in.astronomy.
or. ophthalmology. in. the. sense. that. it. relies. on. wavefront. sensing. and. implementing. a. change. in. the.
active.element.to.correct.for.the.distorted.wavefront..Of.all.the.methods.of.microscopy,.the.two.most.
successful. methods. are,. perhaps,. the. laser. scanning. confocal. luorescence. microscope. and. the. laser.
scanning.two-photon.luorescence.microscope.[1]..Because.both.methods.build.up.an.image.by.scan-
ning.the.excitation.beam.over.the.object,.the.underlying.principles.of.image.formation.are.similar.in.
both.cases;.the.implementation.of.AO.is.likewise.similar.in.both.the.methods..In.this.chapter,.we.focus.
on.the.use.of.a.Shack-Hartmann.wavefront.sensor.(SHWS).for.the.detection.of.relected.excitation.light.
in.the.implementation.of.an.AO.two-photon.microscope.
On.the.one.hand,.two-photon.excitation.luorescence.microscopy.is.a.widely.used.and.popular.method.
of.imaging.biological.samples.as.the.long.wavelengths.used.for.excitation.ofer.deeper.penetration.into.
the. sample. compared. to. single-photon. luorescence. excitation.. On. the. other. hand,. imaging. through.
most.biological.tissue.sufers.from.scattering.and.the.refractive.index.throughout.the.tissue.is.inhomoge-
neous..his.leads.to.distortions.of.the.wavefront.of.the.excitation.light.and.thereby.results.in.an.aberrated.
focus,.reduced.two-photon.eiciency,.and.reduced.imaging.depth..In.fact,.much.of.the.aberrations.that.
are.present.in.imaging.biological.tissue.are.due.to.sample.aberrations.[2-4]..he.causes.of.these.aberra-
tions. are. very. similar. to. the. problems. experienced. in. astronomy. and. ophthalmology. and,. as. with. the.

299