Biomedical Engineering Reference
In-Depth Information
the.primary.determinants.of.aberrations.afecting.the.inal.image.quality..Typically,.the..luorescence.
signal. is. detected. and. changes. are. made. to. the. deformable. mirror. till. the. signal. is. optimized.. In.
this.situation,.the.relected.excitation.light.is.detected..Steps.are.therefore.required.to.eliminate.the.
relected.signals.that.occur.from.planes.that.are.out.of.focus..his.is.accomplished.through.employ-
ing. a. confocal. detection. path. before. the. wavefront. sensor.. As. with. a. conventional. confocal. micro-
scope,.the.confocal.pinhole.allows.only.the.signal.originating.from.the.focal.region.while.efectively.
blocking.the.backscattered.light.emanating.from.other.planes..In.theory,.the.depth.selectivity.(axial.
resolution). should. be. optimized. by. selecting. a. small. pinhole. [10,11].. However,. this. requirement. is.
a.trade-of,.because.the.pinhole.also.functions.as.a.spatial.ilter..Selecting.a.very.small.pinhole.will.
therefore.ilter.out.higher.spatial.frequencies.of.the.distorted.wavefront,.and.in.turn.afect.the.accu-
racy. of. the. wavefront. reconstruction,. which. is. needed. for. optimal. adaptive. correction.. hese. two.
conlicting.requirements.must.be.balanced,.and.the.optimization.of.the.pinhole.size.is.discussed.in.
the.following.section.
16.2.4 overall instrument coniguration
Figure.16.1.describes.the.general.experimental.setup..he.laser.source.was.a.Ti-sapphire.laser.(Tsunami;.
Spectra-Physics,. Mountain. View,. California). pumped. by. a. frequency-doubled. Nd:YVO4. laser.
(Millennia. V;. Spectra-Physics).. he. excitation. light. was. relected. by. the. Mirao52d. deformable. mir-
ror.positioned.at.a.conjugate.plane.to.the.back.aperture.of.the.microscope.objective,.along.the.excita-
tion.optical.path.of.a.typical.two-photon.laser.scanning.microscopy..he.emission.luorescence.signal.
passing. a. short-pass. dichroic. mirror. and. a. short-pass. barrier. ilter. (650dcxxr. and. E700SP;. Chroma.
Technology,. Rockingham,. VT). was. incident. on. a. photomultiplier. detector. (R7400P;. Hamamatsu,.
Detector
Scanner
mirror
8
10
9
Dichroic
mirror
f4
f4
Wavefront
camera
Re
�
ected
beam from
sample
7
13
12
11
6
4
5
3
f4
f4
Sample
Beamsplitter
50/50
Deformable
mirror
Laser
1
2
FIGuRE 16.1
System.coniguration..he.components.used.are.a.Ti-sapphire.(Ti-Sa).laser,.plano-convex.lens.L1.
(
f
=.50.mm),.L2.(
f
.=.200.mm),.L3.(
f
.=.100.mm),.L5.(
f
.=.200.mm),.L6.(
f
.=.400.mm),.L7.(
f
.=.50.mm),.L8.(
f
.= 20 mm),.
444232,.Zeiss.eyepiece,.L10.(
f
.=.125.mm),.L11.(
f
.=.200.mm),.L13.(
f
.=.100.mm),.silver-coated.mirrors,..beamsplitter,.
deformable. mirror. (Mirao52d;. Imagine. Optics),. scanning. mirrors. (6350;. Cambridge. Technology),. short-pass.
dichroic. mirror. (650dcxxr;. Chroma. Technology),. objective. lens. (Fluar,. 1.3. NA;. Zeiss),. photomultiplier. detector.
(R7400P;.Hamamatsu),.pinhole.(P150S;.horlabs),.and.wavefront.camera.(HASO32;.Imagine.Optics).
