Biomedical Engineering Reference
In-Depth Information
animals.at.increasingly.high.sensitivity.by.employing.improved.detection.methods.and.
color-coded. host. tumor. models.. However,. genetic. tagging. is. time-consuming,. complex,.
and.frequently.less.eficient.and.stable.than.alternative.methods,.particularly.when.trying.
to.tag.primary.cells..Bioluminescent.tags,.when.used.as.genetic.probes,.simplify.in.vivo.
detection;.however,.their.signal.is.oxygen.limited,.making.it.dificult.to.determine.whether.
site-to-site.intensity.differences.are.due.to.varying.numbers.of.cells.or.varying.lumines-
cence.eficacy..Most.of.the.genetic.tags.do.not.allow.a.distinction.between.parental.cells.
and.progeny,.and.so.it.is.not.possible.to.determine.the.extent.of.clonal.expansion,.and.it.
can.be.dificult.to.quantify.the.fraction.of.administered.cells.reaching.their.targets.
Carboxyluorescein.succinimidyl.ester.(CFSE),.which
was.originally.developed.for.lym-
phocyte.labeling.and.to.track.their.migration.within.animals.for.many.months,.is.a.luo-
rescent.cell.staining.dye.
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.Subsequent.studies.revealed.that.CFSE.can.react.rapidly.with.
membrane. and. intracellular. proteins. under. physiologic. conditions,. and. can. be. used. to.
monitor.cell.proliferation,.both.in.vitro.and.in.vivo,.due.to.the.progressive.halving.of.CFSE.
luorescence. within. daughter. cells. following. each. cell. division.
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. Despite. these. advan-
tages,.since.CFSE.randomly.labels.various.cell.proteins,.it.can.alter.the.cell.function.due.
to. reaction. with. speciic. functional. proteins.
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. pH. dependence. of. the. carboxyluorescein.
luorescence.intensity.is.another.problem.with.this.probe,.which.can.complicate.analysis.
of.intensity.distributions,.as.alterations.in.subcellular.distribution.pH.may.lead.to.changes.
in.intensity.that.may.be.misinterpreted.as.cell.division..Additionally,.cell.death.causes.the.
same.misinterpretation.due.to.loss.of.intensity.
Lipophilic.luorescent.dyes,.such.as.membrane.dyes,.label.the.lipid.regions.of.cell.mem-
branes,.rather.than.proteins,.and.cause.less.alteration.in.cell.functions..PKH26,.as.a.lipo-
philic.dye,.because.of.its.excellent.membrane.retention.and.biochemical.stability,.has.been.
used. as. the. probe. of. choice. for. in. vivo. tracking. of. ex. vivo-labeled. stem. cells. and. tumor-.
or. antigen-speciic. lymphocytes.. Like. CFSE,. lipophilic. luorescence. intensities. are. halved.
with.each.successive.generation.in.dividing.cells..However,.it.remains.stable.for.weeks.to.
months.in.nondividing.cells..In.addition.to.different.signal.strengths.due.to.dye.redistri-
bution.after.phagocytosis,.these.dyes.have.other.limitations..As.the.membrane.dyes.bind.
noncovalently,.exposure.of.labeled.cells.to.solvents.that.extract.lipids.may.alter.the.dyes'.
intensity,.and.this.should.be.considered.when.using.standard.tissue.ixation.and.process-
ing.methods..A.related.limitation.of.lipophilic.membrane.dyes.is.that.obtaining.reproduc-
ible.results.requires.greater.precision.of.dilution,.because.increasing.dye.concentration.or.
decreasing.cell.concentration.(or.cell.size).results.in.increasing.numbers.of.dye.molecules.
incorporated.per.cell.
Despite. the. widespread. applications. of. luorescence. and. bioluminescence. in. bioimag-
ing,.many.critical.parameters,.such.as.tissue-to-detector.geometry,.autoluorescence,.tis-
sue.optical.properties,.absorption,.and.scattering,.remain.unaccounted.for.in.the.current.
state.of.data.analyses..Furthermore,.organic.luorophores.suffer.from.fast.photobleaching.
and. broad,. overlapping. emission. wavelengths,. and. therefore. are. limited. in. applications.
involving. long-term. imaging. and. multicolor. detection.. Advances. in. synthesis. and. bio-
functionalization.of.colloidal.semiconductor.nanocrystals,.which.are.called.quantum.dots.
(QDs),. during. the. past. decade. have. generated. increasingly. widespread. interest. among.
investigators.in.the.ields.of.biology.and.medicine..Quantum.dots.are.robust.luorescence.
emitters.with.size-dependent.emission.wavelengths..Their.extreme.brightness.and.resis-
tance. to. photobleaching. enable. the. use. of. very. low. laser. intensities. over. extended. time.
periods,.making.them.especially.useful.for.cell.tracking.
