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Author:Väkeväinen, Aaro
Title:Electron-beam induced optical superresolution in integrated light-electron microscopy
Optinen superresoluutio käyttäen hyväksi elektronisuihkua integroidussa valo-elektronimikroskopiassa
Publication type:Master's thesis
Publication year:2014
Pages:vi + 101      Language:   eng
Department/School:Teknillisen fysiikan laitos
Main subject:Optiikka   (F3004)
Supervisor:Törmä, Päivi
Instructor:Hoogenboom, Jacob
Electronic version URL: http://urn.fi/URN:NBN:fi:aalto-201411123033
OEVS:
Electronic archive copy is available via Aalto Thesis Database.
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Location:P1 Ark Aalto  2443   | Archive
Keywords:fluorescence microscopy
correlative light-electron microscopy
optical superresolution
electronbleaching
fluoresenssimikroskopia
korrelatiivinen valo-elektronimikroskopia
optinen superresoluutio
elektronisuihkuvalkaisu
Abstract (eng):We have developed an optical superresolution method based on electronbleaching of fluorophores in integrated light-electron microscopy.
The main advantage of this novel superresolution method is that the non-fluorescent ultrastructure of the sample can be revealed by the simultaneously acquired SEM image.
Furthermore, as the fluorescence superresolution image is based on an electron-beam-induced modification of the specimen, by "switching off" fluorescent probes, both the fluorescence and SEM image are recorded with perfect spatial overlap - being a great advantage for correlative imaging.
The superresolution method is demonstrated with fluorescent microspheres, having a diameter of 40--50~nm.
Their bleaching behaviour is studied as a function of various exposure parameters, and we show that the bleaching rate is mostly dependent on the injected electron dose and electron landing energy.
The superresolution experiments are performed in an integrated light-electron microscope platform (SECOM, Delmic), with which fluorescence emission of the sample can be monitored while the electron beam scans over it.
The method is successfully demonstrated with the fluorescent beads on ITO-coated glass and TEM-grid substrates.
We have achieved a localization precision of approximately 100~nm of the fluorescent beads, and an image resolution of 160~nm -- well beyond the diffraction limit of light.
The method may eventually provide an excellent tool for researchers doing correlative light-electron microscopy in modern life sciences, such as cell and molecular biology.
Abstract (fin):Olemme kehittäneet optisen superresoluutiomenetelmän, joka perustuu fluoresoivien molekyylien elektronisuihkuvalkaisuun integroidussa valo-elektronimikroskopiassa.
Tämän aivan uudenlaisen superresoluutiomenetelmän suurin etu on, että näytteen ei-fluoresoiva hienorakenne pystytään paljastamaan samaan aikaan otetun elektronimikroskooppikuvan avulla.
Koska fluoresenssikuva kerätään "sammuttamalla" fluoresoivia molekyylejä elektronisuihkun avulla, on fluoresenssi- ja elektronimikroskooppikuvien koordinaatisto täsmälleen sama, mikä on suureksi hyödyksi ajatellen korreloivaa kuvantamista.

Superresoluutiomenetelmä demonstroidaan fluoresoivien mikropallojen avulla, joiden halkaisija on 40--50~nm.
Ensimmäiseksi mikropallojen sammumiskäyttäytymistä tutkitaan eri valkaisuparametrien funktiona.
Näytämme, että valkaisunopeus riippuu enimmäkseen syötetystä elektroniannoksesta ja elektronien energiasta.
Superresoluutiokokeet toteutetaan integroidulla valo-elektronimikroskooppialustalla (SECOM, Delmic), jossa fluoresenssisignaalia voidaan tarkkailla samalla kun elektronisuihku pyyhkii näytteen pintaa.

Menetelmän osoitetaan toimivan ITO-päällystetyillä lasialustoilla, sekä TEM-hiloilla.
Olemme saavuttaneet n. 100~nm tarkkuuden fluoresoivien mikropallojen lokalisaatiossa, fluoresenssikuvan resoluution ollessa n. 160~nm, joka on reilusti valon diffraktiorajan alapuolella.
Menetelmä voi aikanaan tarjota erinomaisen työkalun tutkijoille, jotka hyödyntävät korreloivaa valo-elektronimikroskopiaa biotieteissä, kuten solu- ja molekyylibiologiassa.
ED:2014-11-16
INSSI record number: 50059
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