Biomedical Engineering Reference
In-Depth Information
wide-ield.imaging,.it.can.be.efectively.reassigned.to.the.location.of.emission.by.constrained.deconvolu-
tion.algorithms.(Swedlow.et.al..1997).
Under. the. conditions. for. which. wide-ield. microscopy. is. best. suited,. aberrations. can. still. be. quite.
serious..As.shown.in.Figure.15.1,.imaging.20.μm.into.a.live.sample.with.an.oil-immersion.lens.causes.
the.peak.intensity.of.the.point.spread.function.(PSF).to.drop.threefold.and.the.width.of.the.PSF.in.the.
axial.direction.to.increase.by.twofold.(Gibson.and.Lanni.1991).
15.1.1 Sources of Aberrations in Microscopy
he. resolution. achieved. by. modern. objectives,. which. reaches. the. limits. of. what. is. possible. with. lens.
optics,.is.inevitably.linked.with.tight.constraints.on.the.specimen.geometry.and.the.optical.properties.
of.the.light.reaching.the.front.lens.of.the.microscope.objective..Typically,.the.design.of.an.objective.uses.
multiparametric.optimization.of.the.optical.variables.for.about.15.single.lenses.within.the.objective.itself,.
and.also.the.optical.path.and.refractive.index.within.the.immersion.oil.and.coverslip.(Figure 15.2)..he.
objective.accepts.a.given.numerical.aperture.(NA).of.the.wavefront.emerging.from.each.point.within.
the.specimen.and.(for.ininity.corrected.objectives).transforms.it.into.a.plane.wavefront..he.purpose.
of.AO.is.to.correct.the.wavefront.for.varying.specimen.geometries..It.must.be.emphasized.that.this.is.
i
ii
Immersion oil
Coverslip
i
iii
ii
iii
Sample (water)
(b)
(c)
(a)
FIGuRE 15.1
Efect.of.depth.aberrations.on.microscope.point.spread.function.(PSF).
FIGuRE 15.2
Zemax. model. of. 60×. oil. immersion. objective. containing. 15. separate. elements. (Fujimoto. and.
Nishiwaki,.2004).
