tomáš bílý, martin Šemro 28. 07. 2011 simultaneous afm and light microscopy of live cells:...
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Tomáš Bílý, Martin Šemro
28. 07. 2011
Simultaneous AFM and light microscopy of live cells: nano- and microworld within one picture.
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Aims of the project
• Imaging of live cells using AFM and LM
• Development of software tools for cross calibration of the two imaging platforms
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•Light microscopy (LM)•Imaging of cells in vivo
•Atomic force microscopy (AFM)•Surface topography of living cells •Atomic resolution•Mapping of force: elasticity, adhesion
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Lung epithelial cell H441
• major player in lung’s ion balance and liquid clearance proceses
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Principle of AFM
• Cantilever with tip• Piezoelectric scanner• Contact mode
feedback
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Image overlay procedure (AFM on LM)
1. Definition of the tip position in cantilever
- minimal fail overlay: 1 px (LM 20x) = 6 px (AFM)- maximal fail overlay 1 px (LM 20x) = 204 800 px (AFM)
2. Overlay of the images based on the shape of the objecta) high resolution topographyb) force volume mapping
3. Overlay images by specific shape of object
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2. Image overlay by shape of an object
A) Shape of the cell is different in AFM and LM
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2. Image overlay by shape of an object
B) Images recorded in the AFM force volume mapping mode
- overlay still problematic, yet slightly better
- Young’s Modulus on Cell 420 Pa – 20 kPa
surroundings 20kPa – 794 kPa
(example - proteins 0.5 GPa, wood 1GPa)
Overlaid picture
(LM and AFM slope)
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3. Image overlay by specific shape of object
• At best nanometer spherical diameter particles– Quantum dots or other nanoparticles (gold)– Precise determination of the centre of an object in
LM and AFM
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We would like to acknowledge to David Kaftan for leading of this project
Thank you for your attention