super resolution microscopy, and why it is important · 2018-11-23 · 11/23/2018 1 6-9-2018 1...
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Super resolution microscopy,and why it is important
Jelmer Cnossen,Quantitative Nanoscopy Lab, DCSC
qnano.nl
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Microscopy
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Biology:• Self repairing• Self assembling• Self replicating• Renewable
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Biology is programmable
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DNA sequencing and synthesis
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But what do we write?
DNA sequencing and synthesis
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Biology is extremely complex
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Microscopy
Light microscopy Electron microscopy
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Resolving power of microscopes
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Diffraction limited microscopy
• Emitted light from sample is diffracted along path to camera.
• Light from a single emitter is spread over an area on the camera
• Defined by Point-Spread-Function (PSF)
• For visible light: dPSF = ~200 nm
N
y
x
dPSF
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Resolving power of microscopes
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Resolving power of microscopes
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Resolving power of microscopes
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Fluorescence microscopy
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…
x
x x
xt2
t1
t3
…
• Fluorescent molecules are randomly blinking on and off
• The PSF model is fitted to each spot on the camera 3D location
3 μm
Localization Microscopy
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Optical abberations
• Distortion of the light path alongthe way introduces abberations
• Can be corrected with adaptiveoptics
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PSF engineering
data
fit
zstage = -0.5 m zstage = +0.5 mzstage = +0.2 mzstage = -0.2 m zstage = 0 m
double-helix PSF(Pavani/Piestun, Opt.Express 2008; Prasad, Opt.Lett. 2013)
• mean # signal photons / image = 2.4 x 103, background = 11 photons/pixel• 175 nm bead, ex/em = 488/520 nm, NA=1.49• all images contrast stretched for visibility
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Light-sheet fluorescence microscopyIlluminate the focal plane only
Multiscale imaging of plant development by light-sheet fluorescence microscopy
Nature Plants, 2018
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• Lattice light-sheet microscopy: Imaging molecules to embryos at high spatiotemporal resolution
• https://doi.org/10.1126/science.1257998
Lattice Light Sheet Microscopy
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Multiple Determinants and Consequences of Cohesion Fatigue in Mammalian Cells
https://www.biorxiv.org/content/early/2018/05/24/240630
Lattice Light Sheet Microscopy
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• Designing various microscope concepts
• 3D printed / aluminium CNC
• Compact, so we can bring them to biologists
• Create new localization methods
• Combine adaptive optics (thick samples), with light sheets
and localization microscopy
DCSC Course: Control for High Resolution Imaging
Contact:
qnano.nl (work in progress)
[email protected] (Carlas Smith, Group leader)
[email protected] (Me)
What do we do?
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Thanks for listening!
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Automated single-molecule imaging in living cellshttps://www.nature.com/articles/s41467-018-05524-7
Automated single-molecule imaging in living cells
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Theory & practice ...
Enhanced photon collection enables four dimensional fluorescence nanoscopy of living systemshttps://www.nature.com/articles/s41467-018-05799-w