1. Biophysics of green algae: Euglena gracilis investigated by atomic force microscopy C. Gruenberger, R. Ritter, I.C. Gebeshuber Institut fuer Allgemeine Physik, Vienna University of Technology, Wien, Austria Austrian Center of Competence for Tribology AC 2 T, Wiener Neustadt, Austria http://www.ille.com

2. Overview Some properties of biological materials Motivation to study algae Introduction to Euglena gracilis AFM of whole cells AFM of cell organelles Conclusions Outlook (algae, biomaterials)

3. Properties of biological material The hydrodynamic, aerodynamic, wetting and adhesive properties of natural materials are remarkable. The results of evolution often converge on limited constituents or principles. For example, the same material component will be found just slightly but effectively varied to obey different functions in the same organism (e.g. collagen occurs in bones, skin, tendons and the cornea).

4. Properties of biological material One smart feature of natural materials concerns their beautiful organization in which structure and function are optimized at different length scales. Natural systems also show a high level of integration: miniaturization whose object is to accommodate a maximum of elementary functions in a small volume, hybridization between inorganic and organic components optimizing complementary possibilities and functions and hierarchy.

5. Properties of biological material Some biomolecules such as amino acids and thereby also proteins are defined in their structure down to the atomic level. They are materials built with molecular precision. In principle, each and every cell, plant, animal and person can be called a nanotechnological wonder. Nowadays, materials scientists have just started to make man-made materials of such precision.

6. Motivation for studying algae interesting material properties of E. gracilis many TEM and SEM and optical microscopy images available, AFM only sparsely (yields information not only on topography but also mechanical properties) AFM of photoreceptor ultimate goal

7. Introduction to E. gracilis green alga, length 20  m-100  m single celled organism typical model organism for plant researchers and simple animal researchers high pressure resistant natural containers (200 bar)

9. Flagellar swelling: Euglena gracilis photoreceptor photoreceptor with single photon sensitivity at room temperature simple two state photocycle  promising building block in biocomputers Scale bar 100nm, © CNR Pisa

10. Scanning Probe Microscopy (SPM) © Mervin Miles Group, Bristol University, UK

11. Atomic Force Microscopy (AFM) © P. Hansma, UCSB

12. Our AFM setup

13. AFM of whole cells in air Sample preparation (simple drying would result in the cells bursting): 10  l cell suspension are put on a glass slide, slow drying of the suspension covered with cover slip, whole cells are left especially on the edges, embedded in concentrated nutrient solution. AFM tapping mode in air

14. AFM of whole cells

15. flagellum reservoir paramylon grain

16. AFM of whole cells

17. Euglena pellicle SEM © http://www.biol.tsukuba.ac.jp/~inouye/ino/e TEM © Peter v. Sengbusch mechanical stability flexible interlocking ridges slide against each other biogenic lubricants

18. AFM of whole cells

19. Preparation protocol for concentrated solution of crystalline cytoplasm parts Cells were put into solvent (4% Triton X-100 solution). When the membranes had dissolved, the cells were put into a pressure bomb (200bar lN 2 ). Nitrogen slowly diffuses into the cells. Then the cells are released to ambient conditions again and explode because of the high internal pressure). Various centrifugation stages follow in order to separate the remaining organelles from the cytosol.

20. © Barsanti et al., 1993, Vision Res. 33(15), 2043-2050

21. AFM of cell organelles: paramylon grain AFM tapping mode, 2.5  m*2.5  m, height scale 353nm

22. AFM of cell organelles : paramylon grain crystalline carbohydrate energy storage membrane bound granules circumventral stripes --> hot topic of scientific discussion structure -->not yet fully resolved

23. AFM of cell organelles: crystalline lipid body

24. AFM of cell organelles : lipid body about 15 lipid bodies per cell natural wax fat storage

25. Conclusion development of preparation method AFM of cell walls AFM of mucus excretion pellicle pores AFM of new surface features AFM of crystalline cell organelles

26. Outlook I: Towards AFM of the photoreceptor The soluton of crystalline parts contains only few photoreceptors. Lipid bodies, paramylon grains and not completely dissolved cell tissue amount for a large portion of the solution. Combination with fluorescence microscopy is needed.

27. Outlook II: Biomaterials in material science Relating structure to function in biomaterials can only be the beginning of promising developments. The thermal and hydrolytic sensitivities of biological materials limit their applicability in many important synthetic materials applications. A real breakthrough requires an understanding of the basic building principles of living organisms and a study of the chemical and physical properties at the interfaces, to control the form, size and compaction of objects.