Biomedical Engineering Reference
In-Depth Information
Microspheres
Injection site
FIGuRE 17.7
Combination.of.a.diferential.interference.contrast.image.and.a.confocal.image.of.injected.micro-
spheres.in.fruit-ly.embryo.40.μm.below.the.surface.of.the.embryo..he.white.bar.in.the.igure.is.10.μm.long.
throughout.the.embryo..Figure.17.7,.a.combination.of.a.diferential.interference.contrast.(DIC).image.
and.a.confocal.image,.both.taken.with.a.Leitz.inverted.photoscope.equipped.with.laser.confocal.imag-
ing.system.(Leica.Microsystems,.Bannockburn,.IL),.illustrates.that.relative.to.the.injection.site.and.the.
embryo.walls,.the.microspheres.have.spread.randomly.inside.the.embryo..A.higher.microsphere.con-
centration.could.also.be.used.as.the.optical.setup.shown.in.
Figure.17.5
.
allows.for.the.laser.to.illuminate.
one.bead.at.the.time..A.bead.concentration.as.high.as.1:100.has.been.used,.and.the.results.show.many.
more.beads.available.in.the.ield.of.view.(FOV).
17.2.2 Wavefront Measurements
A. reference. Hartmann. sensor. image. was. obtained. to. cancel. the. aberrations. introduced. by. the. opti-
cal.setup.and.coverslip..he.reference.image. was.taken.by.imaging. a.single. luorescent.bead.onto.the.
Hartmann. sensor.. he. bead. was. dried. onto. a. glass. slide. and. imaged. with. the. coverslip. and. mount-
ing. media.. he. image. was. processed. to. obtain. the. location. of. the. Hartmann. spots. using. the. cross-.
correlation.centroiding.algorithm.described.earlier..For.each.wavefront.measurement,.a.new.Hartmann.
sensor.image.was.acquired.with.the.sample.prepared.as.described.in.the.
Section.17.2.1
..he.new.mea-
surement. was. then. processed. to. determine. the. displacement. of. the. Hartmann. spots. (slope. measure-
ments).relative.to.the.reference.image.described.earlier..he.slope.measurements.were.inally.processed.
to.obtain.the.wavefront.by.using.an.FFT.reconstruction.algorithm.(Poyneer,.Gavel,.&.Brase.2002)..For.
each. measurement,. the. peak-to-valley. (PV). and. the. root-mean-square. (RMS). wavefront. errors. were.
collected..he.wavefront.function.was.also.expanded.into.the.Zernike's.circle.polynomials.to.determine.
the.relative.strength.of.the.diferent.modes.(Porter.et.al..2006;.Poyneer.2003)..he.Zernike.polynomials.
are.normalized.and.indexed.as.described.by.Porter.et.al..(2006)..he.wavefront.measurements.were.also.
used.to.analyze.the.PSF.by.taking.the.Fourier.transform.of.the.complex.pupil.function:
2
2
λ
π
FT
P x y
( ',
') exp
⋅
i
w x y
( ',
')
.
.
.(17.5)
x
f
y
f
ξ
=
,
η
=
λ
λ
PSF
( , )
x y
=
2
{
}
FT
P x y
( ',
')
ξ
=
0
,
η
=
0
where.
P
.is.one.inside.the.pupil.and.zero.everywhere.else,.
x
′.and.
y
′.are.the.coordinates.at.the.pupil.
plane,.ξ.and.η.are.the.spatial.frequencies.in.the.transform.domain,.
x
.and.
y
.are.the.coordinates.at.the.
