5/11/2015 MICROSCOPIC TECHNIQUES 2. Fluorescence microscopy SPECIAL TECHNIQUES BASED ON FLUORESCENCE MICROSCOPY

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Giles Mitchell
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UNIVERSITY OF PÉCS MEDICAL SCHOOL www.medchool.pte.

microcopy + fluorecence Image formation i baed on viible light (400 800 nm) and the ue of

Advantage: pectral flexibility provided by the pectral variability of fluorophore excellent

$pecial trick built in a fluorecence microcope 1877 Abbe diffraction limit Ernt Abbe, Carl$ development of uper-reolved fluorecence microcopy" How can we improve the reolution of the

development of uper-reolved fluorecence microcopy" How can we improve the reolution of the

TRICKS TO REDUCTETHE THE POINT SPREAD FUNCTION(PSF PSF) CONFOCAL PRINCIPLE EVANESCENT WAVE

1 UNIVERSITY OF PÉCS MEDICAL SCHOOL MICROSCOPIC TECHNIQUES 2 SPECIAL TECHNIQUES BASED ON FLUORESCENCE MICROSCOPY BIOPHYSICS th March Dr. Beáta Bugyi Department of Biophyic Fluorecence microcopy FLUORESCENCE MICROSCOPY light microcopy + fluorecence Image formation i baed on viible light ( nm) and the ue of gla lene. The object i imaged on the bai of it fluorecence emiion. Advantage: pectral flexibility provided by the pectral variability of fluorophore excellent contrat le invaive pecial technique (FRAP, FRET, FLIM) the reolution can be improved by pecial trick built in a fluorecence microcope 1877 Abbe diffraction limit Ernt Abbe, Carl Zei 2014 Nobel prize in Chemitry Stefan Hell, Eric Betzig and William Moerner "for the development of uper-reolved fluorecence microcopy" How can we improve the reolution of the light microcope? can we ecape phyical law? TRICKS TO REDUCTETHE THE POINT SPREAD FUNCTION(PSF PSF) CONFOCAL PRINCIPLE EVANESCENT WAVE EXCIATION 014/ STIMULATED EMISSION SINGLE MOLECULE LOCALIZATION Ernt Abbe memorial, Jena STRUCTURED ILLUMINATION XY lateral, ~200 MULTI PHOTON EXCIATIONT Z axial 22 ~800 Principle: conjugated focal plane Principle: conjugated focal plane 1

kép light ource detector objective WIDEFIELD MICROSCOPY oneplaneinfocu all

objective CONFOCAL MICROSCOPY oneplanei infocu APERTURE(pinhole): patial

izeoftheaperture~1airyunit Airyunit:diameteroftheAirydik object focal

focal plane fluorecence emiion: behind the focal plane object focal focal

widefield confocal lateral, nm 200 200 axial, nm 800 500 3D imaging

magnet.fu.edu/tutorial/opticalectioning/confocalwidefield/index.

reflection TIRFM total internal reflection fluorecence microcopy tarfih

2 kép light ource detector objective WIDEFIELD MICROSCOPY oneplaneinfocu all the plane contribute to the image APERTURE light ource detector APERTURE objective CONFOCAL MICROSCOPY oneplanei infocu APERTURE(pinhole): patial filter conjugated focal plane one plane contributed to the image izeoftheaperture~1airyunit Airyunit:diameteroftheAirydik object focal plane fluorecence emiion: focal plane fluorecence emiion: in front of the focal plane fluorecence emiion: behind the focal plane object focal plane fluorecence emiion: focal plane fluorecence emiion: in front of the focal plane fluorecence emiion: behind the focal plane WIDEFIELD reolution widefield confocal lateral, nm axial, nm D imaging optical lice OBJECT CONFOCAL many 2D image 3D IMAGE 3D imaging Evanecent wave micorcopy Principle: total internal reflection TIRFM total internal reflection fluorecence microcopy tarfih oocyte meiotic diviion chromoome actin microtubule 2

Evanecent wave micorcopy total internal reflection Evanecent wave microcopy

< widefield, in () in () optically le dene (water) 90 ~100 TIRFM () TIRFM

exponentialdecay () 4 B16/F1 melanoma cell actin filament reolution widefield

Principle: depletion by timulated emiion Stimulated emiion microcopy STED

region 2014 Nobel Prize Stefan Hell, Eric Betzig é William Moerner STED laer

molecule localiation microcopy Principle: PSF fit by a gauian function PALM

microcopy reolution widefield STED lateral, nm 200 50 axial, nm 800 500 () 2

in amphibian cell at reolution of 20 nm (red) and ~30 nm (green channel).

3 Evanecent wave micorcopy total internal reflection Evanecent wave microcopy WIDEFIELD! < widefield, in () in () optically le dene (water) 90 ~100 TIRFM () TIRFM inglemoleculeformin, actin (TIRFM) optically dene (immerion oil) () exp ( ) exponentialdecay () 4 B16/F1 melanoma cell actin filament reolution widefield TIRFM lateral, nm axial, nm Stimulated emiion microcopy Principle: depletion by timulated emiion Stimulated emiion microcopy STED timulated emiion depletion microcopy excitation timulated emion depleted region 2014 Nobel Prize Stefan Hell, Eric Betzig é William Moerner STED laer "for the development of uper-reolved fluorecence microcopy" excitation laer 014/ Stimulated emiion microcopy Single molecule localiation microcopy Principle: PSF fit by a gauian function PALM photo-activated localization microcopy STORM tochatic optical recontruction microcopy reolution widefield STED lateral, nm axial, nm () 2 1 STEDimaging howing two color colocalizedrecording of nuclear pore complexe in amphibian cell at reolution of 20 nm (red) and ~30 nm (green channel). The imaging i decribed in BiophyJ 105, L01 - L03, (July 2013). Göttfertet al Confocal and gated STEDimage of Hitone 3 (green) and microtubule (red) in HeLa cell viualized with Chromeo505 and BD HorizonV500, repectively. 3

Single molecule localiation microcopy Principle: PSF fit by a gauian

optical recontruction microcopy Photoregulable fluorophore STANDARD

localiation microcopy reolution widefield ingle molecule localiation

edu/tutorial/uperreolution/palmbaic/indexflah.

excitation ONE ONE-PHOTON MICROSCOPY excitation: 1 photon aborption:

MICROSCOPY excitation: 1 photon aborption: t = 10-15 E = E excited E

4 Single molecule localiation microcopy Principle: PSF fit by a gauian function PALM photo-activated localization microcopy STORM tochatic optical recontruction microcopy Photoregulable fluorophore STANDARD PHOTOACTIVABLE PHOTOCONVERTIBLE Single molecule localiation microcopy Single molecule localiation microcopy reolution widefield ingle molecule localiation lateral, nm axial, nm () 2 Principle: multi photon excitation Principle: multi photon excitation ONE ONE-PHOTON MICROSCOPY excitation: 1 photon aborption: t = E = E excited E ground photon wavelenght: λ ONE ONE-PHOTON MICROSCOPY excitation: 1 photon aborption: t = E = E excited E ground photon wavelenght: λ TWO TWO-PHOTON PHOTON MICROSCOPY excitation: 2 photon aborption: t = E = E excited E ground photon wavelenght: 2*λ t =

ADVANTAGES improved Z reolution improved penetration depth(higher

wavelength lower energy) imaging tiue in live animal: intravital microcopy

intheliverof a livingmoue dextran, hepatociyte FRAP fluorecence recovery

fluorecence recovery after photobleaching PHOTOBLEACHING irreverible

fluorecence recovery after photobleaching BLEACHING: laer NON diadvantage

glucoeoxidae catalae mercaptoethanol) lower expoure time lower excitation

org/wiki/fluorecence_lo_in_photobleaching http://micro.magnet.fu.

5 ADVANTAGES improved Z reolution improved penetration depth(higher wavelenght lower cattering) confocal: µm / two-photon mm le invaive(higher wavelength lower energy) imaging tiue in live animal: intravital microcopy deep-tiue imaging: imaging deeper layer in the ample Bloodflow intheliverof a livingmoue dextran, hepatociyte FRAP fluorecence recovery after photobleaching Principle: photobleaching/fading + diffuion FRAP fluorecence recovery after photobleaching PHOTOBLEACHING irreverible photochemical detruction of the fluorophore due to the excitation FRAP fluorecence recovery after photobleaching BLEACHING: laer NON diadvantage anti-photobleaching medium(pl. glucoeoxidae catalae mercaptoethanol) lower expoure time lower excitation intenity reitant fluorophore advantage FRAP RECOVERY : DIFFUSION + NON FRAP fluorecence recovery after photobleaching FRAP BLEACHING: laer NON RECOVERY : DIFFUSION + NON 5

6 FRAP FRAP Laiet al EMBO Journal 2009 Summary Modern fluorecence microcopy keyword Principle of reolution enhancing technique TIRFM STED Single molecule localization Principle of pecial imaging technique FRAP Recomended web reource /

Confocal Microscopy & Imaging Technology. Yan Wu

Confocal Microscopy & Imaging Technology Yan Wu Dec. 05, 2014 Cells under the microscope What we use to see the details of the cell? Light and Electron Microscopy - Bright light / fluorescence microscopy