Biomedical Engineering Reference
In-Depth Information
15.2.1 Deformable Mirror control
here.are.several.DMs.currently.on.the.market.and.the.best.choice.depends.on.several.factors..he.most.
important.factors.are.likely.to.be.the.mirror.throw.and.the.number.of.actuators,.which.will.determine.the.
maximum.aberration.amplitude.and.the.maximum.spatial.frequencies.that.can.be.corrected..Other.impor-
tant.factors.are.the.DM.diameter.and.the.response.time..Because.the.back.pupil.plane.will.be.magniied.
onto.the.DM,.the.DM.diameter.will.largely.determine.the.size.of.the.microscope;.the.larger.the.DM,.the.
longer.the.focal.length.lenses.are.required..he.response.time.determines.how.fast.the.mirror.shape.can.be.
changed,.an.important.parameter.in.live.imaging.and.focusing.with.the.DM..he.response.time.of.a.piezo-
electric.
z
-stage.used.for.focusing.is.typically.several.milliseconds;.the.response.time.of.a.DM.will.range.from.
hundreds.of.microseconds.for.micro-machined.DMs.to.several.milliseconds.for.membrane-based.DMs.
For.the.DM,.we.chose.the.Mirao52D.from.Imagine.Optic.because.it.is.capable.of.large.displacements.
and.thus.permits.the.correction.of.aberrations.deep.into.the.sample..he.15-mm-diameter.mirror.has.
52.actuators.on.a.square.grid.with.a.2.5.mm.spacing..he.mirror.is.capable.of.a.maximum.displacement.
of.±75.microns.for.the.focus.mode.
Z
0
( )
.and.±8.microns.for.irst-order.spherical.aberration.
Z
( )
..he.
mirror.can.take.the.shape.of.any.Zernike.through.order.four.with.a.root-mean-square.(RMS).wavefront.
error.of.<20.nm.
We.control.the.mirror.by.measuring.the.wavefront.of.the.HeNe.laser.with.the.wavefront.sensor..We.
reference.the.wavefront.to.a.measurement.with.all.actuators.set.to.zero,.and.then.measure.the.wavefront.
on. the. 32.×. 32. element. SHWFS. for. each. of. the. 52. actuators. activated. individually,. yielding. a. 1024.×.
52.matrix..To.set.a.desired.mirror.shape,.we.use.the.standard.singular-value.decomposition.technique.
(Gavel. 2003). to. determine. the. matrix.
S
,. which. will. yield. the. actuator. values. for. a. desired. wavefront..
Typically,.we.retain.the.irst.45.singular.values.
he.manufacturing.process.results.in.a.small.distortion.of.the.mirror.surface.at.each.actuator,.and.
this.array.of.bumps.(“print.through”).difracts.the.light.causing.small.satellite.peaks.around.the.central.
PSF..his.can.be.clearly.seen.in.Figure.15.6,.which.shows.an.in-focus.PSF.on.a.logarithmic.scale.
15.2.2 Simulation of Microscope and Sample conigurations
To.assess.the.microscope.performance,.we.carried.out.ray-tracing.simulations.of.point.sources.at.various.
depths.below.the.coverslip..While.microscope.optical.designs.can.be.evaluated.and.optimized.using.com-
mercial.optical.sotware.programs,.the.efect.of.the.optical.path.within.specimens.cannot.be.readily.simulated.
by.a.discrete.set.of.optical.elements..To.this.end,.we.wrote.ray-tracing.sotware.that.computes.analytically.the.
ray.paths.through.a.volume.with.continuously.varying.refractive.index.represented.by.a 3D.grid.of.refractive.
PSF
3
4.50
2
4.00
1
3.50
0
3.00
-
1
2.50
-
2
2.00
-
3
-
3
-
2
-
1
0
1
2
3
FIGuRE 15.6
Image.of.a.200.nm.yellow-green.luorescent.bead.on.a.logarithmic.scale..Satellite.peaks.caused.by.
the.print-through.of.the.mirror.are.visible..he.maximum.intensity.of.the.satellite.peaks.is.~1%.of.the.central.peak.
