Biomedical Engineering Reference
In-Depth Information
(with.a.lat.DM,. Figure.15.12a,.c , .and . e ) .show.strong.spherical.aberration..Images.taken.with.the.shape.of.
the.mirror.set.by.Equation.15.1.( Figure.15.12b,.d ,.and . f ) .show.a.more.symmetric.PSF,.similar.to.that.just.
below.the.coverslip..Ater.correction,.the.peak.intensity.is.a.factor.of.two.larger.and.the.width.of.the.peak.
in.the.axial.direction.is.signiicantly.smaller,.meaning.better.axial.resolution..he..correction.removes.
the.low-intensity.“pedestal”.of.light.around.the.main.peak.in.the.uncorrected.image.and.returns.it.to.the.
central.peak,.as.can.be.clearly.seen.in.the.logarithmic.scale.image.( Figure.15.12c .a nd. d ). .he.lateral.plane.
full.width.at.half.maximum.(FWHM).of.the.peak.is.not.signiicantly.changed.by.the.correction,.as.seen.
in. Figure.15.12g ..his.is.because.the.depth.aberrations.do.not.increase.signiicantly.the.width.of.the.cen-
tral.peak..he.aberrations.create.a.broad.pedestal.on.which.the.central.peak.sits.( Figure.15.12c .a nd   d ). .
Ater. correction,. the. PSF. is. very. similar. to. that. at. the. coverslip,. which. should. allow. for. much. better.
reconstruction.using. conventional.robust.space-independent.deconvolution.algorithms.. Figure.15.12i .
and .j .s hows.simulations.of.the.PSF.in.the.axial.direction.for.parameters.corresponding.to.the.measured.
data. . Figure.15.12i .s imulates.a.PSF.65.μm.below.the.coverslip.with.the.same.refractive.index.mismatch,.
and. Figure. 15.12j . simulates. a. well-corrected. PSF. at. the. coverslip.. As. can. be. seen. in. Figure. 15.12 , . the.
agreement.is.quite.good..he.main.diference.is.that.in.the.experiment.the.correction.increases.the.peak.
intensity.by.2.05..In.theory,.the.peak.intensity.of.the.corrected.PSF.is.3.75.times.higher.than.that.of.the.
aberrated.PSF..he.diference.is.most.likely.due.to.the.deviation.of.the.objective.from.the.ideal.case.and.
residual.deviations.in.the.mirror.shape.from.the.theoretical.shape.required.to.correct.the.depth-phase.
aberrations..he.print-through.intensity.peaks.all.around.the.PSF.support.this.reason.for.less-than.ideal.
correction.
he.wavefront.before.and.ater.correction,.calculated.by.phase.retrieval.( Section.15.3.5 ) ,.is.shown.in.
Figure.15.13..he.Strehl.ratio.for.the.corrected.wavefront.is.0.77,.indicating.quite.an.acceptable.correction,.
although.the.edge.of.the.wavefront.in.Figure.15.13b.still.shows.approximately.a.wave.of.residual.aberra-
tion..his.is.possibly.due.to.imperfections.in.the.objective..Interferometric.measurements.of.objectives.
have.shown.that.they.can.be.severely.aberrated.near.the.edge.of.the.pupil.(Juskaitis.2007).
he.results.of.correcting.the.spherical.aberration.in.biological.samples.are.less.dramatic.than.those.of.a.
single.bead.because.the.biological.sample.introduces.scattering.as.well.as.other.aberrations.. Figure 15.14 .
shows.uncorrected.and.corrected.lateral.images.23.μm.below.the.coverslip.of.UMUC..bladder-cancer cells.
with.GFP-TRF1-labeled.telomeres..Although.the.intensity.of.the.cell.autoluorescence.is.not.increased.
by. the. correction. of. the. depth. aberrations,. the. GFP-TRF1. signal. is. brighter. by. 60%. ater. subtracting.
the.broad.autoluorescence.signal.( Figure.15.14c ) ..In.wide-ield.microscopy.aberrations.do.not.change.
the. detected. integrated. intensity.. Because. aberrations. spread. out. the. energy. but. do. not. remove. it,. we.
would. not. expect. increased. intensities. on. correcting. the. depth. aberration. in. a. wide-ield. microscope.
from.spatially.broad.sources.such.as.cell.autoluorescence.and.a.thin.luorescent.sheet.(e.g.,.cytoplasmic.
membrane)..However,.the.intensity.of.sharp.features.is.increased.ater.correction.of.aberrations.
Correcting.the.depth.aberration.or.focusing.with.the.DM.corrects.the.shape.of.the.PSF.and.increases.
the. peak. intensity,. but. as. the. sample. becomes. more. complex,. the. increase. in. intensity. becomes. less.
signiicant..his.is.most.likely.due.to.both.the.importance.of.sample-induced.aberrations.and.the.fact,.
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FIGuRE 15.13 Phase.in.back.pupil.plane.calculated.by.phase.retrieval.from.the.uncorrected.PSF.in. Figure.15.12 .
(let).and.the.corrected.PSF.in. F igure . 15.12 . (right).
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