Biomedical Engineering Reference
In-Depth Information
largely.irrelevant,.while.at.greater.depths,.the.overall.signal.is.signiicantly.lower.due.to.the.scattering.
attenuation.of.both.excitation.and.emission.photons..
Figure.16.9g
.
shows.the.percentage.improvement.
based.on.each.pixel.at.80.μm.depth,.as.calculated.from.
Figure.16.8.
.
Comparing.the.degree.of.improve-
ment.on.the.center.versus.the.edge,.no.major.diference.is.observed..hroughout.the.region.where.lu-
orescence. signal. is. generated,. the. improvement. seems. relatively. uniform.. his. might. be. because. the.
aberration.from.the.optical.system.is.much.larger.than.the.aberration.from.the.sample.
16.3.4 Mouse-Heart-Muscle imaging Using Adaptive optics-
compensated two-Photon Microscopy
As. a. second. example,. we. imaged. the. muscular. structures. of. the. heart. of. a. mouse. with. nuclei. detec-
tion.based.on.endogenous.luorescence,.second.harmonic.generation,.and.exogenous.labeling..Tail-vein.
injection. was. performed. with. a. heavily. anaesthetized. mouse. and. nuclei. were. labeled. with. Hoechst,.
while.the.extracellular.matrix.surrounding.the.cells.was.labeled.with.Texas.Red.Maleimide..Ater.stain-
ing,.the.mouse.was.sacriiced.and.the.heart.was.excised,.ixed.in.4%.paraformaldehyde,.embedded.in.
parain,. and. mounted. without. a. coverslip. ater. some. histological. processing.. he. wavelength. of. the.
excitation.light.was.780.nm..he.objective.lens.was.a.40×.oil-immersion.objective.lens..he.emission.
signal.was.iltered.by.a.green.ilter.(535/40,.Chroma.Technology)..he.ield.size.was.120.×.120.μm.cover-
ing.256.×.256.pixels,.and.the.dwell.time.was.40.μs.
Figure.16.10.shows.representative.images.of.the.mouse.heart.at.80.μm.depth.with.and.without.AO.
compensation..he.image.on.the.let.is.the.uncompensated.image.and.the.image.on.the.right.is.the.com-
pensated. image,. with. both. processed. for. background. rejection.. As. in. the. mouse-tongue. experiment,.
adaptive. correction. was. performed. only. at. the. center. pixel. of. the. image. and. the. deformable. mirror.
shape.was.held.constant.throughout.the.diferent.scanned.positions..To.compare.the.signal.intensity.of.
the.two.images,.the.intensity.distributions.for.all.pixels.were.calculated.again..
Figure.16.11a-d
.
shows.
the.histograms.for.the.intensity.distributions.before.and.ater.the.compensation..he.same.trend.was.
observed. as. in. the. mouse-tongue. result.. he. number. of. brighter. pixels. increased. and. the. number. of.
darker.pixels.decreased.for.all.the.imaging.depths.ater.compensation..To.ind.the.improvement.trend.
with.imaging.depth,.the.mean.photon.count.was.calculated.with.background.rejection..
Figure.16.11e
.
shows.the.percentage.improvement.from.the.mean.photon.counts.according.to.image.depth..he.per-
centage.improvement.shows.that.as.the.imaging.plane.went.deeper,.the.improvement.became.larger.in.
general..he.maximum.improvement.of.the.90-100%.intensity.pixels.was.123%.at.80.μm.depth..In.terms.
of.the.increment.of.photon.count,.the.result.at.20.μm.depth.showed.the.greatest.increase.due.to.generally.
higher.signal.levels..At.greater.depths,.the.scattering.of.excitation.and.emission.photons.was.dominant,.
and.the.improvement.due.to.using.AO.became.less.signiicant..In.terms.of.improvement.at.the.center.
compared.to.the.edge,.it.seemed.to.be.uniform,.similar.to.the.mouse-tongue.result.
100
80
60
40
20
0
20
μ
m
20
μ
m
FIGuRE 16.10
(
See color insert.
). Mouse-heart. images. at. 80. μm. depth. without. and. with. AO. compensation,.
respectively.
