Biomedical Engineering Reference
In-Depth Information
Much. remains. to. be. studied. about. HSCs. and. the. microenvironment. that. HSCs. reside. in.. Ex. vivo.
experiments.on.HSCs.are.limited.by.the.incomplete.understanding.of.the.biological.functionality.of.the.
HSCs.and.the.microenvironment..In.vivo.imaging.of.the.HSCs.has.been.explored.by.utilizing.single-.
and.two-photon.scanning.laser.microscopes.[5].
Because.of.optical.aberrations,.the.TPFM.is.limited.by.both.imaging.depth.and.imaging.resolution..
he.presented.results.show.the.imaging.depth.to.a.few.tens.of.micrometers..However,.it.is.desirable.to.
image.through.the.entire.lat.bone,.which.can.be.several.hundreds.of.micrometers.
12.1.1 Advantages of two-Photon Fluorescence
Microscopy in tissue imaging
Compared. to. single-photon. luorescence. microscopy,. the. two-photon. luorescence. imaging. tech-
nique. has. three. major. advantages. to. allow. imaging. at. the. cellular. level. in. deep. tissue.. First,. two-
photon. .luorescence. imaging. provides. intrinsic. sectioning. ability. when. applied. to. thick-tissue.
imaging.. he. two-photon. luorescence. excitation. is. directly. proportional. to. the. square. value. of.
the. power. density. of. the. excitation. light. at. the. focal. volume.. Out-of-focus. luorescence. .excitation.
drops. of. signiicantly. faster. than. that. of. single-photon. excitation;. in. turn,. less. background. is.
.produced.in.the.imaging.
Second,. two-photon. luorescence. imaging. uses. near-infrared. light. for. luorescence. excitation.. he.
near-infrared.light.sufers.signiicantly.less.light.scattering.when.traveling.through.tissues,.compared.to.
visible.light,.ranging.from.400.to.600.nm..Near-infrared.light.ranges.from.800.to.1500.nm.
Both.scattering.and.absorption.contribute.to.light.attenuation.in.tissue.traveling..he.main.absorb-
ers. of. light. in. blood-perfused. tissues. are. oxyhemoglobin. and. deoxyhemoglobin. [8].. heir. absorption.
spectrum.shows.a.minimum.between.700.and.1000.nm.
hird,.the.two-photon.luorescence.imaging.technique.has.less.photobleaching.than.single-photon.
imaging.. Both. single-. and. two-photon. luorescence. imaging. sufer. photobleaching,. a. photochemical.
process.that.destroys.the.luorescence.agents..For.two-photon.luorescence.excitation,.photobleaching.is.
directly.proportional.to.the.time-averaged.square.value.of.power.density.at.the.light.illumination.path..
Photobleaching.is.conined.to.the.vicinity.of.the.focal.volume,.since.photobleaching.drops.quadratically.
below. or. above. the. focal. plane. [9].. However,. for. single-photon. luorescence. imaging,. photobleaching.
occurs.at.all.regions.of.the.light.illumination.path.
12.1.2 two-Photon Fluorescence excitation and emission
A. luorescent. molecule. in. a. simple. form. can. be. described. as. having. two. diferent. energy. states:. a.
ground. state. and. an. excited. state.. A. ground. state. electron. jumps. to. the. excited. state. by. absorbing.
external.energy.such.as.that.carried.by.photons.from.an.illumination.light..When.the.excited..electrons.
shit.back.to.the.ground.state,.they.can.release.energy.by.emitting.luorescence.photons..For.the.single-
photon.luorescence.excitation,.a.lone.photon.carries.enough.energy.to.excite.a.luorescent.molecule,.
resulting. in. the. emission. of. a. luorescent. photon.. Two-photon. excitation. is. based. on. the. idea. that.
two. photons. of. lower. energy. can. excite. a. luorescent. molecule,. resulting. in. the. emission. of. a. luo-
rescent.photon,.typically.at.a.higher.energy.than.either.of.the.two.absorbed.photons..In.general,.the.
.emitted photon.in.two-photon.excitation.has.twice.the.frequency.and.half.the.wavelength.as.that.of.
the.absorbed.photon.
Two-photon. luorescence. excitation. is. a. nonlinear. luorescence. imaging. technique,. which. also.
includes. other. techniques. such. as. three-. or. four-photon. luorescence. excitation,. second-. or. third-.
harmonic.generation,.and.coherent.anti-Stokes.Raman.spectroscopy.(CARS).
he.most.commonly.used.luorescent.agents.such.as.4',6-diamidino-2-phenylindole,.luorescein.iso-
thiocyanate,.green.luorescent.protein. (GFP),.Cy3,.Cy5,.Texas.red,.and.quantum.dots.have.excitation.
