Biomedical Engineering Reference
In-Depth Information
applications.of.FNDs.as.delivery.vehicles.for.transporting.drugs,.genes,.and.vaccines.into.
cells.have.been.clearly.illustrated.by.these.single.particle.tracking.experiments.
25.4.2 Two-Photon Fluorescence imaging and Fluorescence Lifetime imaging
The. luorescence. image. contrast. of. FNDs. in. biological. cells. can. be. improved. by. two-
photon.luorescence.imaging.and.luorescence.lifetime.imaging..It.has.been.demonstrated.
by. Chang. et. al.. that. two-photon. excitation. provides. better. image. contrast. than. its. one-
photon. counterpart,. because. the. former. can. diminish. most. of. the. cell. autoluorescence.
(Chang.et.al..2008b,.Hui.et.al..2010b)..Additionally,.the.excited.volume.is.smaller.due.to.the.
quadratic.dependence.of.the.luorescence.intensity.on.the.light.intensity.and,.thereby,.the.
photodamage.to.cells.is.signiicantly.reduced..Furthermore,.the.infrared.photons.used.in.
two-photon.excitation.have.a.longer.penetration.through.cells.and.tissue.(Weissleder.and.
Ntziachristos.2003)..Hence,.two-photon.microscopy.can.provide.better.optical.sectioning.
of.FNDs.in.thick.tissue,.especially.in.living.organisms.
On. the. other. hand,. Faklaris. et. al.. have. enhanced. the. luorescence. image. contrast. by.
luorescence.lifetime.imaging.and.investigated.the.uptake.of.FNDs.by.HeLa.cells.(Faklaris.
et. al.. 2008).. The. average. luorescence. decay. lifetime. of. (N-V)
−
. in. bulk. diamond. is. 12.ns.
(Batalov.et.al..2008),.whereas.the.lifetime.of.the.same.defects.in.nanocrystals.is.substan-
tially.lengthened.to.20.ns.(Beveratos.et.al..2001)..This.is.due.to.the.large.change.in.refractive.
index.of.the.surrounding.medium.when.going.from.bulk.diamond.to.nanocrystals.(Tisler.
et.al..2009)..The.lifetime.of.the.defect.center,.either.in.the.bulk.or.in.ND,.is.much.longer.
than.that.of.dye.molecules.and.cell.autoluorescence..So,.one.can.isolate.the.FND.emission.
from.the.autoluorescence.background.of.cells.and.tissue.using.various.time-gating.tech-
niques.to.improve.the.image.contrast.
25.4.3 Super-resolution Optical imaging
The.excellent.photostablity.of.the.(N-V)
−
.center.furnishes.an.opportunity.to.perform.super-
resolution.bioimaging.with.stimulated.emission.depletion.(STED).microscopy.(Rittweger.
et.al..2009a)..The.technique.involves.the.application.of.two.laser.beams.at.different.wave-
lengths.. The. main. laser. beam. (typically. a. blue. or. green. laser). brings. the. luorophore. of.
interest.to.its.excited.state,.while.the.second.laser.beam.(typically,.a.red.laser),.i.e.,.the.STED.
beam,. depletes. this. excited. state. via. stimulated. emission.. The. STED. beam. has. a. shape.
of. a. doughnut. at. the. focus. of. the. microscope. objective,. which. excites. the. outer. region.
around. the. focus.. During. imaging,. the. luorescence. of. the. emitter. excited. by. the. main.
laser.(with.a.regular.Gaussian.beam.proile).stays.unaffected.in.the.center.of.the.doughnut.
spot.but.diminishes.at.the.outer.ring..So,.the.excited.spot.at.the.focus.becomes.apparently.
smaller.than.the.diffraction.limit..Compared.to.other.super-resolution.techniques,.STED.
is.straightforward.and.does.not.require.any.mathematical.post-processing..Therefore,.it.is.
most.suitable.for.high-resolution.imaging.of.living.cells.in.real.time.and.three.dimensions..
However,. the. major. hurdle. of. this. technique. is. that. it. requires. the. use. of. highly. photo-
stable.luorophores.to.avoid.rapid.photobleaching.when.irradiated.by.the.STED.laser.
STED. has. been. applied. to. the. detection. of. single. (N-V)
−
. centers. in. bulk. diamond.
(Rittweger.et.al..2009a)..A.remarkably.high.resolution,.down.to.8.nm,.has.been.achieved..
In. a. separate. work,. Hell. and. coworkers. have. also. successfully. acquired. high-resolution.
images.of.35.nm.FNDs.spin-coated.on.a.glass.plate.using.the.same.technique.(Han.et.al..
2009)..They.obtain.a.resolution.of.∼40.nm,.essentially.limited.by.the.size.of.the.particles..No.
photobleaching.is.found.even.under.the.intensive.STED.laser.irradiation.(up.to.160.mW)..
