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Lucia Gardini LOT/2019 LENS Fluorescence microscopy - - PowerPoint PPT Presentation
Lucia Gardini LOT/2019 LENS Fluorescence microscopy - - PowerPoint PPT Presentation
Lucia Gardini LOT/2019 LENS Fluorescence microscopy Single-molecule fluorescence microscopy Information on distributions and time trajectories that would otherwise be hidden Identify and compare sub-populations Probe biological
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Fluorescence microscopy
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Single-molecule fluorescence microscopy
ONE MOLECULE AT A TIME
Information on distributions and time trajectories that would otherwise be hidden Identify and compare sub-populations Probe biological macromolecules and provide informations on their structure and function In vitro and in vivo
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NA n D d
S
2 sin 2 22 . 1
2
= =
PSF and image resolution
Abbe formula (x, y) Abbre forluma (z) 2/NA2 Radius of x,y PSF Axial amplitude of the PSF
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d d = /2NA
Rayleigh criterion
Lateral resolution ~ 250 nm Axial resolution = 2/NA2 ~ 500nm
Image resolution
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~ 250 nm
Overcoming the diffraction limit
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x y x centre y centre
Localized point
Fluorescence Imaging with One Nanometer Accuracy FIONA
Thompson et al. Biophys.J. 2002
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Fluorescence Imaging with One Nanometer Accuracy FIONA
Thompson et al. Biophys.J. 2002
b N
?
High quantum yield fluo probes High NA aperture
- bjectives (1.45)
High sensitivity CCD cameras such as EMCCD
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Yildiz et al. Science 2003
High quantum yield fluo probes High NA aperture
- bjectives (1.45)
High sensitivity CCD cameras such as EMCCD
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LOCALIZATION ON SURFACES:TIRF MICROSCOPY
100 nm depth in the sample Small volume excited High S/N
100 nm
TOTAL INTERNAL REFLECTION MICROSCOPY (proximity to the membrane)
b
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LOCALIZATION ON SURFACES:TIRF MICROSCOPY
INCLINED ILLUMINATION HILO (Highly inclined and Laminated Optical sheet)
THE ILLUMINATION BEAM ALWAYS PASSES THROUGH THE CENTER OF THE SPECIMEN PLANE ALLOWING OPTICAL SECTIONING 8 FOLD HIGHER SIGNAL/BACKGROUND COMPARED TO TRADITIONAL WIDEFIELD MICROSOCPY
b
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Yildiz et al. Science 300:2061, 2003
Application in vitro: myosin V walks hand over hand
Accuracy: 1.5 nm. Time resolution: 0.5 s 10^4 photons collected
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Singel Molecule FRET (Forster Resonance Energy Transfer)
Distanze tipiche tra 30 e 80 Å
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R = dist.donor/acc R0 = dist.caratteristica (50% di en. trasferita) Es.Cy3/Cy5 R0= 60 Å (6 nm)
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Probing conformational changes and displacements
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Lucia Gardini 09/04/2019 LENS
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NOBEL PRIZE IN CHEMISTRY 2014
Xiaowei Zhuang BREACKTHROUGH PRIZE 2019
Single molecule localization microscopy: SUPER-RESOLUTION MICROSCOPY
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The principle of PALM and STORM
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The principle of PALM and STORM
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FIONA
The principle of PALM and STORM
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The principle of PALM and STORM
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The principle of PALM and STORM
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The principle of PALM and STORM
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The principle of PALM and STORM
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The principle of PALM and STORM
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The principle of PALM and STORM
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The principle of PALM and STORM
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The principle of PALM and STORM
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The principle of PALM and STORM
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Activation Imaging laser (657 nm) Activation laser (532 nm) Cy3 Cy5 Cy3 Cy5 Cy3 Cy5
20 15 10 5
Activation laser pulses Cy5 fluorescence Time (s)
Activator Reporter 6000 photons
STORM - Photo-switchable Probes
More colors
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Direct STORM
Dempsey G.T. et al Nat. Methods 2011
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5 μm B-SC-1 cell, Microtubules stained with anti-β tubulin Cy3 / Alexa 647 secondary antibody
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5 μm
Bates et al, Science 317, 1749 – 1753 (2007)
106 molecules, 2-30 min
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500 nm
5 μm
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5 μm █ Cy3 / Alexa 647: Clathrin █ Cy2 / Alexa 647: Microtubule
Bates et al, Science 317, 1749 – 1753 (2007)
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1 μm
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200 nm
Avg = 172 nm
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3D via astigmatic detection
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10 nm lateral 20 nm axial
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