Biomedical Engineering Reference
In-Depth Information
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FIGuRE 17.23
Wavefront.measurements.using.YFP-labeled.structures.as.guide.stars.in.mouse.brain.tissue..he.
excitation.light. source.is.focused. on.a.dendrite.in.(a).and.on.a.cell. body.of.the. neurons.in.(b)..he.crossed. lines.
indicate.the.location.of.the.focus.point.for.the.wavefront.measurements..he.wavefront.measurements.using.a.YFP-
labeled.structure.is.shown.for.a.dendrite.in.(c).and.a.cell.body.in.(d)..he.scale.bar.is.5.μm.
he.measurement.errors.for.diferent.numbers.of.photons.per.subaperture.can.be.measured.using.the.
method.described.in.Morzinski.et.al..(2010)..he.EM.gain.setting.for.the.EMCCD.in.SHWF.sensor.was.
200×..he.photons.per.subaperture.for.the.wavefront.measurements.shown.in.Figure 17.23c.and d.are.
1950.and.2047,.which.produce.0.03λ.in.measurement.error..his.amount.of.measurement.error.is.small.
and. improvements. at. this. level. will. also. depend. on. the. errors. caused. by. limitations. of. the. DM. and.
wavefront. temporal. variations.. he. RMS. wavefront. errors. shown. in. Figure 17.23c. and. d. are. 0.1742λ.
and.0.3471λ.(λ.=.527 nm),.respectively..Photobleaching.is.tested.for.the.YFPs.in.a.dendrite,.because.it.
has.a.lower.concentration.of.luorophores.and.thus.photobleaches.more.quickly..Confocal.images.were.
obtained.every.30.seconds.during.the.exposure.of.the.laser.with.23.nW.at.the.back.aperture.of.the.objec-
tive.lens.for.wavefront..sensing..he.intensity.change.in.the.focus.area.is.shown.in
.
Figure.17.24.
.Ater.
the.irst.three.minutes,.the.intensity.drops.less.than.10%..Considering.the.exposure.time.(500.ms).for.
the.wavefront.measurement,.photobleaching.caused.by.the.wavefront.measurement.is.very.limited..For.
live. samples,. higher. laser. power. can. be. applied. because. the. luorescence. recovers. ater. photobleach-
ing..herefore,.shorter.exposure.times.can.be.achieved..he.line.proile.along.two.subapertures.on.the.
SHWF.sensor.image.is.shown.
he. confocal. images. for. the. sample. are. collected. by. scanning. along. the.
z
-axis. with. a. 3.μm. range.
and.a.0.15.μm.step.size..he.inal.images,.as.shown.in.
Figure.17.25
,
.are.achieved.using.the.maximum.
intensity.projection.applied.to.the.images..Ater.turning.on.the.wavefront.correction.loop,.the.wavefront.
error.converges.ater.10.iterations,.which.takes.0.30.seconds..he.YFP.on.the.cell.body.was.used.as.a.
guide-star,.which.is.located.at.a.depth.of.70.μm,.as.shown.in.Figure.17.23b..Before.correction,.the.RMS.
wavefront.error.is.0.3471λ..Ater.correction,.the.measured.wavefront.error.is.0.03λ.RMS,.as.shown.in.
Figure. 17.25e
.
. he. confocal. images. before. correction. and. ater. correction. are. shown. in.
Figure. 17.25a
.
and
.
b
..
he.intensity.proile.along.the.dashed.lines.across.a.dendrite.and.a.spine.is.shown.in
.
Figure.17.25f
.
.
he.intensity.increased.by.3×..he.image.of.the.dendrite.and.the.spines.are.much.clearer.ater.correction.
with.improved.image.contrast.
We.also.tested.the.fruit-ly.embryos.labeled.with.GFP-polo.and.EGFP-Cnn.(Tao.et.al.,.2012)..To.test.
the.ability.of.the.wavefront.correction.at.a.deep.depth,.the.confocal.images.with.and.without.corrections.
