Biomedical Engineering Reference
In-Depth Information
A.data.acquisition.board.(National.Instruments,.TX).is.synchronized.with.raster.scanning..mirrors.to.
feed.the.PMT.signal.to.the.AO.loop..he.typical.AO.speed.is.1.Hz..A.Pentium.4.IBM.compatible.com-
puter.is.used.to.control.the.DM.
he.optical.ilters.are.selected.to.accommodate.the.absorption.and.emission.spectrums.of.the.GFP..
GFP. has. a. relatively. wide. absorption. spectrum.. he. absorption. spectrum. peaks. at. 488. nm,. and. the.
luorescence. emission. peaks. at. 509. nm.. In. the. two-photon. process,. the. illumination. wavelength. will.
be.920 nm..To.match.the.GFP.model,.the.chosen.dichroic.ilter.is.a.long-pass.laser.transmitting.ilter.
(Semrock,.NY)..he.transmitting.window.is.from.750.to.1100.nm,.and.the.relecting.window.is.from.
350  to. 720. nm.. An. optical. band-pass. ilter. (Semrock,. NY). was. also. installed. in. front. of. the. PMT. to.
eliminate.the.photon.noise..he.transmission.spectrum.for.the.band-pass.ilter.is.from.400.to.700.nm.
12.5 experiments and results
12.5.1 Point Spread Function Quantiication
he. intrinsic. three-dimensional. imaging. ability. from. the. TPFM. provides. a. signiicant. advantage. in.
tissue. imaging.. he. efects. of. AO. on. a. TPFM. can. be. demonstrated. by. the. change. of. the. PSF. of. con-
structed.TPFM.with.and.without.AO.correction..AO.is.used.to.correct.both.the.inherent.optical.system.
and.the.sample.aberrations.
he. PSF. deines. the. optical. resolution. of. a. microscope,. and. it. can. be. measured. based. on. the. light.
intensity.distribution.at.the.focal.volume..he.PSF.from.a.TPFM.can.be.approximated.by.a.Gaussian.
function. both. laterally. and. axially.. he. full. width. at. half. maximum. (FWHM). of. the. itted. Gaussian.
function. is. then. a. good. measure. of. the. optical. quality. of. a. TPSL-FM.. he. theoretical. FWHM. under.
difraction-limited.conditions.for.the.planned.TPFM.are.0.37.μm.laterally.and.1.4.μm.axially..he.illu-
mination.light.wavelength.is.0.92.μm.
To.measure.the.excitation.light.intensity.distribution.of.the.focal.volume.from.a.TPFM,.it.was.neces-
sary.to.design.a.scanning.system.that.had.sub-micrometer.imaging.resolution..A.“simpliied”.version.of.
the.TPFM.with.AO.was.developed.for.this.purpose,.as.shown.in.Figure.12.9..A.single.tip-tilt.scanning.
Translation stage: Z
scan
Samples
Ti-sapphire
laser
Objective
Dichroic
filter
Deformable
mirror
Tip-tilt mirror
PMT
Computer/data
acquisition
FIGuRE 12.9 he.two-photon.scanning.laser.microscope.setup.for.PSF.measurement..he.tip-tilt.mirror.is.used.
for.the.x-y.scan,.and.translation.stage.is.used.for.the.z.scan..he.line.in.dark.shade.marks.the.illumination.beam..
he.line.in.pale.shade.indicates.the.luorescence.signal.
 
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