Biomedical Engineering Reference
In-Depth Information
Camera
Wavefront sensor
Excitation light
from fiber
f
ex
1
f
sh
2
f
2
f
ex
2
f
sh
1
Dichroic 1
Dichroic 3
Dichroic 2
60x oil
objective
Tube lens
f
1
`
Reference beam
FIGuRE 15.5
Microscope.layout..See.text.for.details.
where.
M
. is. the. objective. magniication.. Lens.
f
1
. is. chosen. as. 350. mm. so. that. the. NA. of. the. system. is.
1.285..To.collect.light.from.a.sample.below.the.coverslip,.the.maximum.NA.is.set.by.the.sample.refrac-
tive.index,.implying.for.aqueous.media.NA.<.1.33,.in.any.case..An.NA.of.1.285.in.water.corresponds.to.a.
maximum.angle.of.74°,.greater.than.the.70°.maximum.angle.for.an.NA.of.1.42.in.immersion.oil.(1.512);.
the.lower.NA.does.not.correspond.to.a.less-challenging.imaging.coniguration..Lens.
f
2
.=.800.mm.was.
chosen. so. that. the. sample. is. Nyquist. sampled. on. the. CCD. camera.. his. microscope. uses a .camera.
from. Astronomical. Research. Cameras,. Inc.. (San. Diego,. CA). with. a. cooled. CCD57-10. chip. from. e2v.
Technologies.(Chelmsford,.UK)..he.pixel.size.is.13.microns..For.Nyquist.sampling,.we.require
λ
M
f
f
.
2
1
>
d
.
.(15.6)
pixel
4
NA
For.emission.at.510.nm,.the.Nyquist.pixel.size.is.99.nm..And.for.the.component.values.chosen.here,.
the.efective.pixel.size.at.the.sample.plane.is.94.6.nm..he.additional.components.between.the.tube.lens.
and.the.CCD.camera.reduce.the.light-collecting.eiciency.of.the.system.by.no.more.than.22%..his.loss.
could.be.minimized.by.using.dielectric.mirrors.instead.of.metallic.mirrors.
he.excitation.light.at.488.nm.(Coherent.Innova).passes.through.an.optical.shutter.(UniBlitz).and.is.
focused.onto.a.rotating.difuser.(Physical.Optics).to.remove.spatial.coherence..he.light.is.then.coupled.
into.a.multimode.iber.(600.μm.core,.0.22.NA;.Ocean.Optics)..Lenses.
f
ex1
.and.
f
ex2
.magnify.the.iber.end.
onto.the.DM;.because.the.DM.is.in.the.back.pupil.plane,.this.is.a.Kohler.illumination.coniguration..
Dichroic.1.(Semrock.FF495-Di01).inserts.the.excitation.light.at.488.nm.into.the.beam.path.and.sends.
the.emission.light.(510.nm).to.the.CCD.camera.
he.mirror.shape.is.monitored.with.a.Shack-Hartmann.wavefront.sensor.(SHWFS;.Haso.32;.Imagine.
Optic,.Orsay,.France).that.measures.a.light.beam.from.a.632.nm.HeNe.laser.inserted.into.the.optical.
path.ater.the.objective.lens.with.a.notch.ilter,.dichroic.2.(Semrock.FF.579/644-Di01),.and.sent.to.the.
SHWFS.ater.the.DM.with.another.notch.ilter,.dichroic.3.(Semrock.FF.579/644-Di01)..Lenses.
f
sh1
.and.
f
sh2
.demagnify.the.beam.to.image.the.DM.onto.the.lenslet.array.
Placing.the.DM.in.a.conjugate.plane.to.the.back.pupil.guarantees.that.the.same.wavefront.correction.
is.applied.over.the.entire.ield.of.view.
