Biomedical Engineering Reference
In-Depth Information
Microscopes,.irst.developed.around.1650,.have.been.essential.to.the.progress.of.biological.science..
Every. incremental. improvement. in. microscope. resolution. has. been. followed. by. a. wave. of. indings. in.
biology. and. a. deeper. understanding. of. life.. In. 1655,. Hooke. built. his. compound. microscope. leading.
immediately.to.the.description.of.cells..In.1674,.Leeuwenhoek.built.his.simple.microscopes.from.glass.
beads. and. described. protozoa. and. bacteria.. Improvements. in. transmitted. light. microscopy. allowed.
Brown. to. describe. the. nucleus. in. orchids. in. 1833,. and. Schleiden. and. Schwann. to. state. cell. theory. in.
1838..Following.Abbe's.work.in.1876.on.increasing.microscope.apertures,.Flemming.described.mitosis..
Cajal.used.stains.to.see.tissue.anatomy.in.1881.and.Koch.launched.microbiology.in.1882..Following.Zeiss.
and.Abbe's.building.of.a.difraction-limited.microscope.in.1886,.Golgi.described.the.Golgi.apparatus.
(1898).. Following. the. discovery. of. radioactivity. by. Curie,. Lacassagne. developed. autoradiography. in.
1924..Lebedef's.interference.microscope,.followed.by.Zernike's.phase-contrast.technique,.allowed.sci-
entists.to.see.cells.migrating.out.of.a.biopsy.into.tissue.culture.dishes.and.to.develop.cell.lines..Coone's.
development.of.the.luorescent.microscope.(1941).and.Ploem's.(1975).implementation.of.epiluorescence.
enabled.immunoluorescence.imaging.of.the.molecular.constituents.of.cells..Today,.microscopy.is.the.
central.tool.for.following.complex.spatiotemporal.processes.in.cells,.embryos,.and.whole.animals..he.
development.of.genetic.labeling.with.green.luorescent.protein.(GFP).and.its.variants.ofers.a.direct.link.
between.the.list.of.genes.encoding.the.molecular.components.of.life.and.their.functions.
Microscopy. was. originally. developed. to. image. thin,. two-dimensional. samples,. and. modern. high-
resolution.luorescence.microscopes.are.optimized.to.create.difraction-limited.images.of.the.plane.just.
below.the.coverslip..But.biological.samples.are.three.dimensional.(3D),.and.microscopy.has.increasingly.
been.used.over.the.past.three.decades.to.develop.3D.luorescence.views.by.moving.the.focal.plane.of.the.
microscope.objective.through.the.depth.of.the.sample.(Agard.and.Sedat.1983;.Agard.et.al..1988;.Agard.
et.al..1989).
In.3D.microscopy,.as.the.imaging.plane.moves.deeper.into.the.sample,.the.image.quality.becomes.
degraded. because. of. optical. aberrations. and. scattering.. Both. scattering. and. optical. aberrations. are.
caused.by.refractive-index.variations..Scattering.is.the.result.of.submicron.inhomogeneities.in.the.tissue.
and.can.best.be.treated.as.multiple.scattering.events.in.the.sample.that.lead.to.a.randomized.phase.and.
amplitude.distribution.of.the.light..Aberrations.are.caused.by.larger-scale.variations.in.the.refractive.
index.that.lead.to.a.distortion.of.the.light.wavefront.(Tuchin.2007).
For.imaging.ixed.biological.samples.with.luorescence.microscopy,.sample-induced.aberrations.can.
be.reduced.or.eliminated.by.optical.clearing.(Sullivan.et.al..1999;.Tuchin.2007),.and.depth.aberrations.
can.be.eliminated.by.matching.the.index.of.the.ixation.medium.with.that.of.the.immersion.oil.(Staudt.
et.al..2007)..But.for.live.3D.imaging,.the.correction.of.aberrations.is.more.critical.because.the.sample.
index.cannot.be.manipulated..A.water-immersion.objective.can.be.used.to.more.closely.match.the.index.
of.a.live.sample..However,.this.solution.cannot.correct.for.refractive.index.variations.within.the.sample.
and.only.pushes.the.point.at.which.depth.aberrations.become.serious.from.a.few.microns.to.a.few.tens.
of.microns..AO.promises.to.correct.aberrations.allowing.high-resolution.3D.live.imaging.
Today.there.are.multiple.microscopy.modalities.using.transmitted.light.(phase.contrast.and.digital.
image. correlation. [DIC]),. epi-illumination. (dark. ield. and. luorescence),. and. nonlinear. optical. tech-
niques.(multiphoton.luorescence,.Raman.scattering,.second.and.third.harmonic.imaging,.and.others)..
All.these.techniques.would.beneit,.in.principle,.from.AO..Because.luorescence.microscopy.is.such.a.
central.tool.in.cell.biology,.much.of.the.work.on.AO.in.microscopy.to.date.has.focused.on.this.tech-
nique..In.particular,.the.emphasis.has.been.on.confocal.and.multiphoton.microscopy,.techniques.that.
are.intended.for.deep.imaging.in.tissue.samples.
Although.wide-ield.microscopy.loses.contrast.more.signiicantly.with.depth.as.compared.to.confo-
cal. or. multiphoton. microscopy,. there. are. many. conditions. under. which. it. is. the. preferred. technique..
Murray.et.al..(2007).demonstrated.that.wide-ield.microscopy.is.the.most.sensitive.luorescence.micros-
copy.technique.under.low-scattering.conditions..Furthermore,.live.biological.samples.are.very.sensitive.
to.the.light.excitation.dose.(Carlton.et.al..2010)..hus,.wide-ield.luorescence.microscopy.is.well.suited.
to.in.vivo.imaging.in.samples.where.scattering.is.not.too.large..Although.out-of-focus.light.is.present.in.
