Biomedical Engineering Reference
In-Depth Information
TABlE 17.2
Isoplanatic.Angle.Measurements.for.the.40×.Magniication,.0.75.NA.Objective.Lens
1
14
10.7
1.60
1.90
0.73
2
18
13.8
0.98
1.24
0.77
3
25
19.1
1.69
1.10
1.30
Mean
19
±
5.57
14.5
±
4.25
1.42
±
0.39
1.41
±
0.43
0.93
±
0.32
where.φ.is.the.wavefront.in.radians,.
X
.is.a.vector.representing.the.two-dimensional.(2D).coordinates,.θ
0
.
is.the.isoplanatic.angle,.and.σ
θ
2
.is.the.mean-square.error.between.the.measured.and.observed.wavefront..
We.can.determine.the.isoplanatic.half-width.by.multiplying.the.isoplanatic.angle.by.the.focal.length.of.
the.objective.
To.determine.the.isoplanatic.angle,.we.took.wavefront.measurements.from.two.microspheres.separated.
by.a.distance.
d
..A.microsphere.was.excited.by.shining.a.laser.on.it..Each.microsphere.was.excited.indi-
vidually..Each.wavefront.sensor.measurement.was.collected.over.a.period.of.500.ms,.much.longer.than.the.
typical.AO.loop.bandwidth..his.ensures.that.there.is.little.noise.on.the.data..he.standard.deviation.for.
each.individual.wavefront.was.measured.to.better.than.1%.of.the.wavelength.at.647.nm..Table.17.2.shows.
three.diferent.measurements.taken.with.a.40×.(0.75.NA).objective.lens..he.irst.measurement.shows.that.
the.wavefront.error.for.the.bead.located.at.the.center.of.the.FOV.RMS(1).is.1.60.radians,.the.wavefront.
error.for.the.bead.located.14.μm.from.the.center.RMS(2).is.1.90.radians,.and.the.wavefront.diference.
between.the.two.measurements.RMS(1.−.2).is.0.73.radians..Taking.the.average.of.three.measurements.
shows. that. the. isoplanatic. half. width. is. 19.±. 5.6.μm.. hese. results. show. that. a. reference. microsphere.
together.with.an.AO.system.can.help.improve.the.quality.of.the.images.taken,.not.just.at.the.location.of.
the.microsphere.but.also.within.a.circle.20.μm.in.radius.
17.2.5 Wavefront corrections and Fluorescent
imaging at Different Wavelengths
We.have.taken.wavefront.measurements.at.one.wavelength.(red).that.have.been.used.to.make.correc-
tions.to.luorescently.labeled.beads.at.a.diferent.wavelength.(green)..Green.beads.were.used.to.emulate.
biological.structures.that.are.labeled.with.green.luorescent.protein.(GFP).using.well-known.structures.
that.have.a.size.(1.μm).that.is.near.the.difraction.limit.of.the.optical.system.
he.images.in.
Figure.17.14
.are.of.green.luorescent.beads.that.were.excited.using.the.488.nm.laser.and.
imaged.with.the.green.science.camera.(Azucena.et.al..2011)..In.
Figure.17.14a
,.the.AO.system.is.of,.and.
the.mirror.was.put.on.a.lat.position.that.had.been.calibrated.using.an.interferometer..he.lat.position.
is.kept.on.regardless.of.correction.on.or.of.to.reduce.systematic.aberrations..We.can.see.some.details.
about.structures.20.μm.below.the.surface.of.the.embryo,.but.we.are.not.able.to.resolve.the.individual.
beads.that.make.up.the.clumps.of.material.shown.in.the.image..In
.
Figure.17.14b
,.the.AO.system.had.
been.turned.on,.and.we.can.clearly.resolve.the.individual.1.μm.luorescent.beads.
.
Figure.17.14c
.
and.
d.
show.cross-sectional.proiles.along.the.gray.lines.in
.
Figure.17.14a
.
and
.
b
,
.respectively..hese.igures.show.
that. with. the. AO. system. on,. we. can. clearly. resolve. the. individual. beads. and. thus. are. able. to. obtain.
higher.resolution.structural.information..Even.though.the.wavefront.aberrations.were.measured.using.
the.crimson.beads,.the.corrections.applied.to.the.mirror.still.improve.the.image.of.the.green.luorescent.
beads,.which.are.more.than.100.nm.apart.in.wavelength.
17.3 Ao confocal Fluorescence Microscope
he.principle.of.the.confocal.microscope.is.shown.in
.
Figure.17.15
.
.A.patent.on.this.invention.was.iled.
in.1957.by.Marvin.Minsky.and.was.issued.in.1961.(Minsky.1961)..he.focal.plane.of.a.coherent.light.
from.a.laser.source.is.conjugate.with.the.pinhole.located.in.front.of.the.detector..he.light.is.relected.by.
a.dichromatic.mirror.and.focused.on.the.sample.through.an.objective.lens..he.emission.light.from.the.
