Biomedical Engineering Reference
In-Depth Information
5
Specimen-Induced
Geometrical Distortions
5.1.
Introduction.and.Overview...............................................................75
5.2.
Problem.Description...........................................................................75
5.3.
Experimental.Setup.............................................................................77
5.4.
Relation.between.Zernike.Aberration.Modes.and.
Geometric.Distortion.........................................................................77
5.5.
Results.and.Discussion.......................................................................78
Michael Schwertner
confovis GmbH
5.6.
Summary.and.Conclusion................................................................. 81
5.1 introduction and overview
Specimen-induced.aberrations.afect.the.imaging.properties.in.optical.three-dimensional.(3D).micros-
copy,.especially.when.high-numerical-aperture.(NA).lenses.are.used..In.Chapter.4,.we.have..measured.
aberrations.to.understand.their.efect.toward.image.quality.parameters,.such.as.intensity.and..resolution..
Apart. from. these. efects,. aberrations. can. also. introduce. geometric. image. distortions,. a. fact. that. is.
oten. overlooked.. his. is. because. it. cannot. be. observed. directly. in. the. reciprocal. epi-.coniguration.
of. a. .common. luorescence. microscope.. Still. the. efect. of. specimen-induced. distortions. can. lead. to.
.substantial.errors.for.spatial.measurements.
In.this.chapter,.transmission-mode.interferometric.data.are.used.to.quantify.the.geometric..distortions.
associated.with.the.specimen-induced.aberrations..his.information.is.otherwise.inaccessible.but.allows.
the.estimation.of.the.accuracy.of.spatial.measurements.within.acquired.3D.data.sets..An.assessment.for.
a.range.of.biological.specimens.shows.that.spatial.measurements.can.be.signiicantly.compromised.by.
the.specimen-induced.aberrations.
5.2 Problem Description
In. an. ideal. microscope,. spherical. wavefronts. converge. toward. an. optimum,. difraction-limited. focal.
spot.at.the.nominal.focal.position.(NFP)..Unfortunately,.the.specimen.under.examination.can.introduce.
deviations.from.this.ideal.wavefront,.which.lead.to.an.aberrated.focal.spot..he.efect.of.these..aberrations.
can.be.divided.into.two.categories..he.irst.category.produces.a.blurred.and.enlarged.focal.spot,.which.
leads.to.a.loss.in.resolution.and.image.intensity..his.efect.is.caused.by.the.Zernike.aberration.modes.
higher.than.4.(see.Table.4.1.of.Chapter.4),.and.this.is.investigated.in.detail.in.the.same.chapter.
he.second.category.is.investigated.in.this.chapter..he.associated.efects.are.related.to.the.three.lower-
order.Zernike.modes:.2.(tip),.3.(tilt),.and.4.(defocus)..hey.are.not.aberrations.in.the..classical.sense.and.
have.no.inluence.on.the.intensity.distribution.of.the.focal.spot.but.create.a..displacement.between.the.
actual.focal.position.(AFP).and.the.NFP.as.shown.in.
Figure.5.1
..A..standard.3D.microscope.system.maps.
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