Biomedical Engineering Reference
In-Depth Information
group. velocity. dispersion. caused. by. the. ive. lenses. (including. objective). in. the. sample. arm,. several.
prisms.(GVD-P).are.placed.in.the.reference.arm..A.right-angle.prism.on.a.linear.translation.stage.allows.
modiication.of.the.reference-arm.length.to.match.the.sample.arm.without.changing.the.direction.of.
the.beam..In.some.cases,.additional.attenuation.of.the.reference.arm.was.desired,.which.is.realized.using.
another.neutral.density.ilter.(ND2)..Phase.shiting.was.realized.using.a.piezo-mounted.folding.mirror.
(M6/PZT).. Note. that. in. contrast. to. the. simple. CGWS. sample. described. earlier,. the. reference. arm. is.
not.folded.back.onto.itself.in.this.implementation,.but.is.rejoined.with.the.light.coming.back.from.the.
sample.by.means.of.a.second.(nonpolarizing).beam.splitter.(BSC1).
To.avoid.overcomplicating.the.setup,.no.scanning.mirrors.(which.need.to.be.conjugated.to.the.BFP.
and.therefore.would.require.another.telescope.in.the.sample.arm).were.integrated..Instead,.
x
,.
y
,.and.
z
.
scanning.were.performed.by.moving.the.sample.using.a.piezo-driven.translation.stage.(TS1a).
For.two-photon.imaging,.a.dichroic.mirror.(M
Dichr
).separated.the.backscattered.excitation.light.(for.
CGWS).and.the.luorescence.light.(for.imaging)..he.latter.was.collected.with.a.lens.(L7).onto.a.photo-
multiplier.tube.(PMT),.ater.rejection.of.residual.transmitted.excitation.light.by.means.of.an.absorptive.
blue-green.ilter.(F).
14.5.2 in Vivo Adaptive correction
Using. the. setup. described. earlier,. anesthetized. transgenic. zebra. ish. (
Danio rerio
). larvae. with. GFP.
interneurons. were. imaged. with. and. without. wavefront. correction. (Rueckel. et. al.. 2006).. he. larvae,.
which.had.been.chemically.treated.to.inhibit.pigment.formation,.were.embedded.in.agarose.and.placed.
under. the. microscope. objective. to. allow. access. to. the. olfactory. bulb. (see. Figure. 14.9a);. imaging. was.
1
µ
m
0
µ
m
-
1
µ
m
(a)
(b)
133
225
0
0
10
µ
m
10
µ
m
(c)
(d)
FIGuRE 14.9
(a). Experimental. setup:. an. anesthetized. zebra. ish. embedded. in. agarose. is. placed. under. a. two-
photon.microscope.with.adaptive.optics.for.imaging;.(b).optimal.wavefront.for.a.focus.position.inside.the.olfactory.
bulb.(OB),.50.μm.below.the.surface.of.the.head;.(c).two-photon.image.of.neurons.in.said.position.in.the.OB,.with-
out.adaptive.optics;.(d).two-photon.image.of.the.same.region.ater.pre-compensation.of.the.aberrations.shown.in.
panel.b..From.Rueckel,.M.,.J..A.,.Mack-Bucher,.et.al..(2006)..Adaptive.wavefront.correction.in.two-photon.micros-
copy. using. coherence-gated. wavefront. sensing..
Proc. Natl. Acad. Sci. USA
.
103
(46):17137-17142.. Copyright. 2006.
National.Academy.of.Sciences,.USA.
