Biomedical Engineering Reference
In-Depth Information
TABLe 16.1
Comparison.of.AFM.with.Other.Microscopic.Techniques
AFM
LM
TEM
SEM
Resolution
1-2.nm
100-200.nm
1-2.nm
5-10.nm
Contrast
Very.high.
(no.staining)
Medium.(no.staining).
or.high.
(staining.required)
High.(staining.
required)
High.(staining.
required)
Interior/surface
Surface.only
Both
Interior
Surface.only
Sample.
preparation
Needs.to.be.adhered.
to.substrate;.no.
ixing,.staining.
required;.near-
physiological.
conditions
No.need.for.adherence;.
staining.required.for.
contrast.enhancement;.
near-physiological.
conditions
Dehydrated,.ixed.
and.stained.
samples.on.TEM.
grids
Dehydrated,.ixed.
and.metal-coated.
samples.on.SEM.
stubs
Additional.
capabilities
Force.measurement;.
nanomanipulation
Fluorescence
Energy.dispersive.
spectroscopy;.
diffraction
Energy.dispersive.
spectroscopy;.
backscattered.
electron.detection
Successful. AFM. imaging. critically. depends. on. a. strong. and. stable. adherence. of. the.
sample. to. a. substrate.. This. is. because. in. most. imaging. modes,. the. AFM. probe. comes.
into. physical. contact. with. the. sample. and. can. potentially. push. around. unbound.
molecules..In.most.cases,.the.puriied.biological.sample.is.immobilized.onto.a.substrate.
by. means. of. electrostatic. attractions. or. covalent. bonding,. making. the. substrate. type,.
sample. concentration,. selection. of. sample. buffers,. and. washing. procedures. important.
considerations.when.preparing.single-molecule.samples.for.AFM..The.substrate.of.choice.
is. usually. mica. because. it. can. be. easily. cleaved. to. generate. a. fresh,. negatively. charged,.
molecularly.lat.surface..Glass,.silicon,.and.cell-culture.surfaces.are.also.common.substrates.
because.they.can.be.functionalized.to.ensure.a.covalent.chemistry.with.biological.samples.
and/or. nanoparticles.. The. pH. and. ionic. composition. of. the. selected. sample. buffers. can.
also.impact.the.surface.charge.of.the.molecules.and.either.hinder.or.enhance.their.ability.
to.immobilize.on.the.substrates..Finally,.the.washing.and.drying.procedures.necessary.for.
removing.loosely.immobilized.molecules.may.vary,.depending.on.the.degree.of.sample-
substrate. afinity. and. the. environment. (i.e.,. air. or. luid). desired. for. imaging.. Careful.
considerations. for. each. of. these. components. can. signiicantly. impact. AFM. imaging..
Details. of. biological. sample. preparations. required. for. AFM. have. been. reviewed. in. the.
literature.(El.Kirat.et.al..2005).
16.3.1 Single-Molecule imaging
In. the. ield. of. nanobiotechnology,. AFM. is. most. popularly. utilized. as. a. characterization.
tool.for.single-molecule.imaging.of.biomolecules.and.nanoparticles..The.ability.to.image.
not. only. in. air. but. also. in. liquid. environments. makes. the. AFM. especially. attractive. for.
single-molecule.imaging.applications.because.aqueous.solutions.tend.to.best.preserve.the.
structural. integrity. of. biological. samples.. The. AFM. has. produced. topographs. of. DNA.
fragments.(Gudowska-Nowak.et.al..2009),.plasmid,.and.even.supercoiled.DNA.(Tanigawa.
and.Okada.1998).in.both.air.and.aqueous.environments.(Lyubchenko.and.Shlyakhtenko.
1997)..Nanoparticle.and.nanotube.samples.of.various.materials,.sizes,.and.geometries.have.
also. been. characterized. in. air. (Rao. et. al.. 2007). and. luid. (Baer. et. al.. 2010).. Additionally,.
