1. Tomáš Bílý, Martin Šemro 28. 07. 2011 Simultaneous AFM and light microscopy of live cells: nano- and microworld within one picture.

2. Aims of the project Imaging of live cells using AFM and LM Development of software tools for cross calibration of the two imaging platforms

3. Light microscopy (LM) Imaging of cells in vivo Atomic force microscopy (AFM) Surface topography of living cells Atomic resolution Mapping of force: elasticity, adhesion

4. Lung epithelial cell H441 major player in lung’s ion balance and liquid clearance proceses

5. Principle of AFM Cantilever with tip Piezoelectric scanner Contact mode feedback

6. 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 object a) high resolution topography b) force volume mapping 3.Overlay images by specific shape of object

7. 2. Image overlay by shape of an object A) Shape of the cell is different in AFM and LM

8. 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)

9. 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

10. We would like to acknowledge to David Kaftan for leading of this project Thank you for your attention