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1、Fast,long-term,super-resolutionimagingwithHessianstructuredilluminationmicroscopyToincreasethetemporalresolutionandmaximalimagingtimeofsuper-resolution(SR)microscopy,wehavedevelopedadeconvolutionalgorithmforstructuredilluminationmicroscopybasedonHessianmatrixes(He
2、ssian-SIM).Itusesthecontinuityofbiologicalstructuresinmultipledimensionsasaprioriknowledgetoguideimagereconstructionandattainsartifact-minimizedSRimageswithlessthan10%ofthephotondoseusedbyconventionalSIMwhilesubstantiallyoutperformingcurrentalgorithmsatlowsignalin
3、tensities.Hessian-SIMenablesrapidimagingofmovingvesiclesorloopsintheendoplasmicreticulumwithoutmotionartifactsandwithaspatiotemporalresolutionof88nmand188Hz.Itshighsensitivityallowstheuseofsub-millisecondexcitationpulsesfollowedbydarkrecoverytimestoreducephotoblea
4、chingoffluorescentproteins,enablinghour-longtime-lapseSRimagingofactinfilamentsinlivecells.Finally,weobservedthestructuraldynamicsofmitochondrialcristaeandstructuresthat,toourknowledge,havenotbeenobservedpreviously,suchasenlargedfusionporesduringvesicleexocytosis.
5、Recentyearshavewitnessedanexponentialgrowthintheuseofsuper-resolution(SR)fluorescencemicroscopy,providingnewmechanisticinsightsintomanybiologicalprocesses1–4.However,becausethelightdoserequiredforSRimagingisusuallymarkedlyhigherthanthatusedinconventionalmicroscopy
6、,ithaslongbeenrecognizedthatlive-cellSRmicroscopytechniquesareseverelylimitedbyphotobleachingandphototoxicity5.Therefore,forcellbiologystudies,ultrafastSRmicroscopytechniquesandlong-termlive-cellSRmicroscopystrategiesremaintobedeveloped.Thedevelopmentofbrighterand
7、morephotostabledyesisoftenregardedasacrucialsteptowardlong-termSRimaging6,7.However,thefluorescenttaggingofcellularproteinsatahighdensitymightnotalwaysbepossible,andthehighilluminationintensityusedforphotostabledyesmightinteractwithendogenouschromophorestogenerate
8、photodamage5.Thefluorescenceemissionoffluorescenttagsalsosaturatesathighexcitationpowers,forexample,at~1.5kW/cm2forEGFP8.Simplyincreasingtheillumination
