Biomedical Engineering Reference
In-Depth Information
x-y
x-y
x-y
13,000
(a)
(b)
(c)
Water-Immersion
objective
9,750
6,500
3,250
Brain slice
Water
0
x-z
y-z
x-z
y-z
x-z
y-z
Slide
(d)
Full correction
System correction
15,000
10,000
5,000
0
0
1
2
3
4
5
6
Distance (
µ
m)
4
4
4
(e)
3
3
3
2
2
2
System
correction
System
correction
(4x)
Full
correction
1
1
1
0
0
0
FIGuRE 13.10
Correction.of.aberrations.induced.by.250.μm.thick.ixed.mouse.brain.slices..(a-c).Lateral.(
x
-
y
).
and.axial.(
x
-
z
.and.
y
-
z
).images.of.a.single.1.μm.diameter.bead.under.a.brain.slice,.with.only.system.correction.(a),.
with.a.4.×.display.gain.(b).and.ater.full.AO.correction.(c)..(d).Lateral.intensity.proiles.along.the.light.gray.and.dark.
gray.lines.in.(a).and.(c)..(e).he.corrective.wavefront,.ater.subtraction.of.system.aberrations,.in.units.of..excitation.
light. wavelength. (850. nm),. using. 12. independent. subregions. and. direct. phase. measurement.. Scale. bars:. 2. μm..
(Ji, N.,.et al.,.
Nat. Meth
.,.7,.2,.141-147,.2010.)
biological. applications,. it. would. be. desirable. if. the. intrinsic. luorescence. from. the. sample,. rather.
than. a. guide. star. like. a. luorescent. bead,. can. be. used. for. AO. correction.. Instead. of. using. the. cen-
troid. shit. of. a. point. object,. such. as. the. luorescent. bead,. to. measure. the. beam. delection,. one. can.
use. image. correlation. to. measure. the. image. shit. when. diferent. pupil. segments. are. turned. on.. In.
Figure.13.11
,
.luorescent.images.of.several.neurons.at.the.bottom.of.a.ixed.brain.slice.were.used.to.
measure.the.aberration.caused.by.a.cover.slip.and.the.300-μm-thick.brain.tissue..hree.sets.of.image.
data. are. shown—without. correcting. the. system. aberration,. with. system. aberration. correction,. and.
with.correction.for.both.the.system.and.sample-induced.aberration—and.the.image.quality.improves.
progressively.. (It. is. not. always. true. that. correcting. the. system. aberration. improves. image. quality..
Depending.on.the.nature.of.the.aberrations,.system.aberration.may,.in.some.cases,.partially.cancel.
the. sample-induced. aberration.. As. a. result,. correcting. the. system. aberration. may. actually. reduce.
the. image. quality.). he. improvement. is. especially. striking. in. the. axial. plane,. because. axial. resolu-
tion.is.usually.more.susceptible.to.aberration.than.lateral.resolution—a.long-standing.observation.in.
microscopy.[15].that.can.be.simply.explained.with.our.focus.model..For.most.of.the.light.rays.within.
the.excitation.beam,.as.the.rays.start.to.deviate.from.and.miss.the.focus,.the.volume.they.form.has.a.
longer.dimension.axially.than.laterally.
In.addition.to.ixed.in.vitro.samples,.AO.can.be.used.to.improve.imaging.quality.in.vivo..Using.the.
pupil-segmentation-based. AO,. we. systematically. characterized. and. corrected. the. optical. aberrations.
