Biomedical Engineering Reference
In-Depth Information
Microspheres
(a)
(b)
FIGuRE 17.18
Wide-ield.microscopy.images.of.mouse-brain.tissue.(thickness.=.100.μm).with.(a).10×.objective.
and.(b).60×.water.immersion.objective..he.focal.plane.is.at.the.bottom.of.the.tissue..he.microsphere.is.deposited.
on.the.glass.slide.
(a)
(b)
FIGuRE 17.19
Confocal.imaging.of.mouse-brain.tissue.without.AO..he.thickness.of.tissue.is.100.μm..(a).he.
image.on.the.top.surface.of.the.sample..(b).he.confocal.image.at.the.bottom.surface.of.the.sample.
diameter.crimson.luorescent.microspheres.(Invitrogen,.Carlsbad,.CA).were.deposited.onto.a.glass.slide.
and. below. a. cover. plate. for. use. as. laser. guide-stars.. Sample. brain. coronal. sections. of. diferent. thick-
nesses.(15,.50,.and.100.μm).were.cut.with.a.microtome..he.tissues.are.mounted.with.antifade.mount-
ing.medium.(Invitrogen).on.glass.slides.coated.with.luorescent.microspheres..Figure.17.18a.shows.the.
image.of.the.brain.tissue.with.a.thickness.of.100.μm..he.microspheres.below.the.tissue.are.shown.in.
Figure.17.18b.
17.3.2.1 confocal imaging without Wavefront compensation
he.spherical.aberration.induced.by.the.cover.glass.is.initially.compensated.by.adjustment.of.a..correction.
collar. on. the. objective. lens.. he. system. aberration. is. further. corrected. by. the. DM. by. .measuring. the.
wavefront. aberration. from. a. microsphere. at. the. bottom. of. the. cover. plate. using. the. SHWF. sensor..
he thickness.of.the.brain.tissue.is.100.μm..he.confocal.imaging.system.scans.from.the.top.surface.to.
the.bottom.surface..he.FOV.is.about.50.μm..he.frame.rate.is.30.frames/second.for.fast.scanning..To.
improve.the.SNR,.300.frames.are.averaged.to.generate.the.inal.image..Figure.17.19a.shows.the.confocal.
image.at.the.top.surface,.where.the.cell.body,.dendrite,.and.spine.can.be.observed..Figure.17.19b.shows.
the.image.at.the.bottom.surface..Because.of.the.aberrations.induced.by.the.tissue,.it.is.very.hard.to.see.
the.dendrites.and.spines.around.the.cell.body.
17.3.2.2 confocal imaging with Wavefront compensation
Brain.tissues.with.thickness.of.15,.50,.and.100.μm.are.imaged.in.the.AO.system..A.motorized.Z.stage.
under.the.sample.focuses.the.HeNe.laser.on.the.microsphere.at.the.bottom.of.the.tissue..he.wavefront.
