Biomedical Engineering Reference
In-Depth Information
the. experimental. result,. the. efective. NA. of. the. objective. was. about. 0.55. in. our. experimental. setup,.
although.its.theoretical.NA.is.1.3,.which.is.valid.only.when.immersion.oil.is.present.under.the..objective.
lens.till.a.coverslip.and.the.sample.are.located.immediately.beneath.and.with.a.refractive.index.of.1.5..
his.current.limitation.can.be.removed.by.utilizing.a.top-hat.beam.proile.or.modifying.the.AO.feedback.
algorithm.to.ignore.information.from.the.peripheral.region.of.the.wavefront.sensor.
16.3.3 Mouse-tongue-Muscle imaging Using Adaptive
optics-compensated two-Photon Microscopy
Mouse-tongue. musculature. was. visualized. based. on. both. endogenous. luorescence. and. second. har-
monic.generation..A.whole.tongue.excised.from.a.female.C57BL/6.mouse.(10.weeks.old).was.ixed.in.
phosphate-bufered. saline. (PBS,. pH. 7.4). containing. 2.5%. glutaraldehyde. for. a. day.. he. ixed. tongue.
tissue.was.then.immersed.in.PBS.for.3.h,.rinsed.with.bufered.solution.repeatedly,.embedded.in.parain,.
and.mounted.without.a.coverslip..he.wavelength.of.the.excitation.light.was.set.to.780.nm..he.objective.
lens.was.a.Zeiss.40×.oil.immersion.objective..he.emission.signal.was.iltered.by.a.green.ilter.(535/40,.
Chroma.Technology)..he.ield.size.was.120.×.120.μm.covering.256.×.256.pixels..he.dwell.time.was.
40 μs..Figure.16.8.shows.representative.images.of.the.imaged.mouse-tongue.muscle.at.80.μm.depth,.with.
and.without.the.AO.compensation..he.image.on.the.let.is.the.uncompensated.image.and.the.image.
on. the. right. is. the. compensated. image,. with. both. processed. for. background. rejection.. he. threshold.
for. each. image. was. set. to. three. times. the. intensity. level. measured. in. the. regions. outside. the. objects.
of.interest.(e.g.,.blood.vessels.in.the.mouse.heart.or.the.neurons.in.the.mouse.brain)..In.principle,.the.
bandwidths.of.the.wavefront.camera.and.the.deformable.mirror.are.about.20.and.200.Hz,.respectively..
However,.the.current.feedback.algorithm.takes.signiicant.CPU.resources.and.leads.to.a.compensation.
time.of.4.s-5.s..he.algorithm.therefore.forms.the.bottleneck.for.the.bandwidth.of.the.compensation.
process..At.these.time.scales,.it.is.far.from.practical.to.perform.pixel-by-pixel.correction.over.the.whole.
image..To.reduce.the.overly.long.compensated-imaging.process,.the.assumption.that.the.aberrations.are.
primarily.due.to.depth.rather.than.ield.position.was.made.[2-4],.and.AO.compensation.was.performed.
only.at.the.center.pixel.of.the.image..his.mirror.shape.was.kept.for.the.rest.of.the.image.acquisition..
In.future.studies,.it.would.be.interesting.to.determine.the.optimal.number.of.pixels.that.should.be.cor-
rected. per. image,. given. the. trade-of. between. imaging. speed. and. tissue. heterogeneity.. It. would. also.
be.interesting.to.improve.imaging.speed.by.developing.faster,.more.eicient.feedback.algorithm.or.by.
utilizing.higher-performance.computer.hardware.
he.compensated.images.show.higher.signal.strength.when.compared.to.the.uncompensated.images..
Figure.16.9a-d
.
shows.the.histograms.for.the.intensity.distributions.before.and.ater.compensation..he.
number.of.brighter.pixels.increased,.while.the.number.of.darker.pixels.decreased.for.all.imaging.depths.
ater. compensation,. which. implies. that. the. in-focus. luorescence. signals. were. being. optimized. while.
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12
10
8
6
4
2
0
20
μ
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20
μ
m
FIGuRE 16.8
(
See color insert.
). Mouse-tongue. images. at. 80. μm. depth. without. and. with. AO. compensation,.
respectively.
