Biomedical Engineering Reference
In-Depth Information
subregions.and.direct.phase.measurement,.the.full.width.at.half.maximum.of.the.bead.images.in.both.
the.lateral.and.axial.directions.approached.their.difraction-limited.values.
13.4.2 nonbiological Samples
Our.pupil-segmentation-based.AO.approach.is.very.efective.when.used.to.correct.optical.aberrations.
caused.by.refractive.index.mismatch.in.nonbiological.samples..In.the.irst.example,.we.used.it.to.correct.
for.the.aberrating.efects.when.the.water-dipping.1.0.NA.Zeiss.objective.was.used.to.image.a.500 nm-
diameter. luorescent. bead. in. air. (Figure. 13.8a-d).. he. pupil. was. segmented. into. 49. subregions,. and.
the phase. correction. was. measured. ater. the. delection. measurement. was. carried. out. on. each. subre-
gion..Compared.to.the.images.taken.only.with.system.aberration.correction,.the.peak.signal.ater.full.
AO.correction.increased.eightfold,.and.both.the.lateral.and.the.axial.full.width.at.half.maximum.val-
ues.approached.their.difraction-limited.values..he.sample-induced.aberration.was.clearly.dominated.
by. spherical. aberration,. with. a. peak-to-valley. value. of. 20. wavelengths.. Furthermore,. the. correction.
remained.valid.over.a.98.×.98.μm.ield.of.view.[9]..In.other.words,.we.transformed.this.water-dipping.
objective.into.an.air.objective..With.AO.correction,.one.can.now.use.the.same.objective.in.immersion.
media.of.diferent.refractive.indices.by.simply.applying.the.corresponding.corrective.wavefront.to.the.
SLM..In.another.example,.we.imaged.a.500.nm.diameter.bead.placed.on.the.inside.surface.of.a.glass.
capillary. tube. that. was. immersed. in. water.. he. bead. positioned. well. away. from. the. centerline. of. the.
capillary.tube.yielded.a.highly.asymmetric.wavefront,.which.could.nevertheless.be.corrected.by.pupil.
segmentation..Ater.such.correction,.the.signal.increased.about.3.5-fold,.and.near-difraction-limited.
26,800
20,100
13,400
6,700
0
x
-
y
16,000
12,000
8,000
4,000
0
x
-
y
(a)
x
-
y
(b)
x
-
y
(e)
(f)
Water-immersion
objective
Water-immersion
objective
x
-
z
x
-
z
x
-
z
x
-
z
Capillary
tube
Air
Slide
(c)
Water
30,000
30,000
Slide
System correction
Full correction
System correction
Full correction
(g)
25,000
25,000
System correction
Full correction
System correction
Full correction
15,000
15,000
15,000
20,000
20,000
15,000
15,000
10,000
10,000
10,000
10,000
5,000
5,000
5,000
5,000
y
-
z
y
-
z
0
0
0
1
2
3
0
10
20
30
40
0
0
Distance (
µ
m)
Distance (
µ
m)
0
1
2
0 1 2 3 4 5 6 7 8 9 10
Distance (
µ
m)
Distance (
µ
m)
20
(d)
(h)
5
16
12
4
3
8
2
1
System
correction
Full
correction
4
System
correction
Full
correction
0
0
FIGuRE 13.8
Correction. of. aberrations. in. nonbiological. samples.. (a. and. b). Lateral. and. axial. images. of. a. 500.
nm. luorescent. bead. viewed. in. air. using. a. water-dipping. objective,. with. correction. for. only. system. aberrations.
(a).or.all.aberrations.(b)..(c).Signal.proiles.in.the.lateral.(
x
-
y
).and.axial.(
x
-
z
).planes.along.the.solid.and.dashed.
lines.in.(a) and.(b)..(d).he.inal.corrective.wavefront.in.units.of.wavelength.(850.nm),.ater.subtraction.of.system.
aberrations,.obtained.with.49.independent.subregions.and.direct.phase.measurement..(e.and.f).Images.of.a.bead.
in.an.air-illed.capillary.tube.in.water,.with.system.aberration.correction.only.(e).and.full.correction.(f)..(g).Signal.
proiles.in.the.lateral.(
x
-
y
).and.axial.(
x
-
z
).planes.along.the.solid.and.dashed.lines.in.(e).and.(f)..(h).he.inal.correc-
tive.wavefront.in.units.of.excitation.wavelength.(850.nm),.ater.subtraction.of.system.aberrations,.obtained.with.36.
subregions.and.direct.phase.measurement..Scale.bars:.2.μm..(Ji,.N.,.et.al.,.
Nat. Meth
.,.7,.2,.141-147,.2010.)
