Biomedical Engineering Reference
In-Depth Information
TABLE 15.1
Comparison.of.AFM,.SEM,.and.TEM.Imaging.Methods
AFM
TEM
SEM
Max..resolution
Subnanometer
Atomic
Nanometer
Typical.cost.(US$)
100,000-300,000
500,000.or.higher
200,000-400,000
Imaging.environments
Air,.luid,.vacuum,.
controlled.temperature
Vacuum
Vacuum
In.situ.imaging
Yes
No
No
Simultaneous.optical.imaging
Yes
No
No
Sample.preparation
Easy
Dificult
Medium
15.5 KeyMethodologicalConsiderationsforAFM
Scanningof
Plasmodium
spp.IRBCs
One.of.the.most.important.considerations.before.conducting.AFM.scanning.is.how.best.to.
prepare.the.sample..On.the.one.hand,.RBCs.are.a.highly.deformable.and.nonadherent.cell.
type..It.is.very.dificult.to.achieve.high.resolution.and.stable.images.directly.from.liquid.
environment.without.any.treatments.on.the.cells.and.substrates..Thus,.it.will.be.helpful.to.
immobilize.the.cells.on.certain.pretreated.substrates.and.reduce.the.cells'.deformability.to.
minimize.the.scanning-induced.movement.of.the.sample..Previously.developed.protocols.
involved. using. a. range. of. ixatives. to. ix. the. cells. to. withstand. the. scanning. force. from.
the.AFM.tip.
11
.However,.such.ixation.methods.may.alter.the.surface.morphology.of.the.
sample.signiicantly.without.the.operator's.awareness..A.recently.developed.nonixation.
smearing.method.helped.to.maintain.the.cells'.structures.at.low.cost.and.labor.
10
On.the.other.hand,.there.are.some.considerations.one.should.be.aware.of.when.preparing.
clinical.samples..First,.blood.collected.from.patients.should.be.conserved.in.anticoagulant.
such.as.lithium.heparin.or.citrate,.not.in.ethylenediaminetetraacetic.acid..(EDTA).which.has.
been.observed.to.cause.signiicant.apoptotic-like.changes.to.the.IRBC,.such.as.membrane.
detachment..As.we.wish.to.focus.on.the.erythrocytes,.it.is.also.better.to.remove.the.leuko-
cytes. contaminating. the. sample,. thus. eliminating. a. messy. artifact-illed. background.. This.
can.be.achieved.with.commercial.ilters.or.simple.self-made.cellulose.ilters.at.a.fraction.of.
the.cost.
12
.It.is.also.important.to.note.that.the.erythrocytic.life.cycle.of.
Plasmodium
.spp..has.
numerous.distinct.stages.(for.example,.asexual.stages.have.ring,.trophozoite,.and.schizont.
forms),.and.depending.on.the.species.of.the.isolate.examined,.the.proportion.of.these.stages.
may.be.signiicantly.skewed..For.example,.most.of.the.stages.present.in.
P. falciparum
.isolates.
collected.from.the.peripheral.blood.are.at.the.ring.or.early.trophozoite.stage,.as.the.mature.
cytoadherent.knob-covered.IRBCs.at.the.trophozoite.and.schizont.stages.have.sequestered.
to.the.microvasculature..Therefore,.if.one.wishes.to.examine.the.knobs.present.on.the.mature.
asexual.form,.the.isolate.must.be.cultured.in.a.nutrient-rich.media.at.37°C.under.microaero-
phillic.conditions.for.20.to.40.h,.depending.on.the.stage.required..Once.the.sample.has.the.
required. stage. present,. it. should. be. washed. twice. in. an. isotonic. solution. before. smearing.
onto.a.good.quality.microscopic-grade.glass.slide..To.more.eficiently.identify.both.the.stage.
and.location.of.IRBCs,.the.sample.could.be.supravitally.stained.with.a.luorescent.DNA.dye.
(such.as.4',6-diamidino-2-phenylindole.(DAPI)).or.a.very.low.concentration.of.Giemsa.(less.
than.2%)..The.smear.should.not.be.too.dilute,.as.the.isotonic.solution.supporting.the.cells.
will.form.large.salt.crystals.on.the.surface.of.IRBCs,.which.will.impede.the.scanning.
