1. Biljana Stojković Mentor: Prof. Dr Igor Poberaj University of Ljubljana Faculty of Mathematics and Physics Ljubljana, December 4th, 2012

2.  Introduction  Microrheology  Optical tweezers  Passive Microrheology  Active Microrheology  Rheology of bacterial network  Future work Outline

3.  Microrheology Rheology is the study of the deformation and flow of a material in response to applied force. materials properties materials properties solid solid fluidfluid VISKOELASTICVISKOELASTIC polymers foams foams bacteria gels DNA  Rheology

4. Applying oscillatory shear strain:

5. Microrheology is »Microscopic probe particles »Locally measure viscoelastic parameters »Study of heterogeneous environments »Requires less than 10 microliters of sample »Biological samples – limited amount of material »Important for fundamental reaserch and in industrial applycations “rheology on the micrometer length scale” Current techniques can be divided into two main categories: active methods that involve probe manipulation passive methods that rely on thermal fluctuations of the probe

6. Technique in microrheology

7.  Optical tweezers technique

8. How we could describe the trapping of dielectric bead?  R<<λ, point dipol  R>>λ, ray optics λ Rλ Rλ Rλ R λ Rλ Rλ Rλ R

9. Optical tweezers set-up

10. Power Spectral Density (PSD): Force calibration

12.  Passive microrheology Brownian motion Two ways for determination shear modulus: 1. 2. Linear response theory:

13.  Active microrheology  One-particle active Oscillations of trap:

14.  The displacements od the probe particle:  Active microrheology  Two-particle active  The same displacements can be also expressed directly as:

15.  Active microrheology Complex viscoelastic modulus: Mutual response functions: Single particle response functions:

16. Rheology of bacteria network Different modes: Free floating mode Formation of biofilms Bacteria – single cell organisms

17. Biofilms Free-floating organisms attach to a surface Colonies of bacteria embedded in an extracellular matrix (EPS) EPS consist of: Polymers and proteins accompanied with nucleic acids and lipids  Protect microorganisms from hostile enviroment  Support cells with nutrients  Allow comunication between cells EPS:

18. Biofilm development Lag phase Log phase Stationary phase Death phase

19. Complexity of biofilm arises: The production and assembly of cells, polymer, cross-links and surfactants result in a structure that is heterogeneous and dynamic. Spatial heterogeneities in extracellular chemical concentration; Regulation of water content of the biofilm by controling the composition of EPS matrix; Spatial heterogeneities on gene expression creates heterogeneities in polymer and surfactant production

20. Why is this study important Biofilm mechanics is important for survival in some enviroments Well-known viscoelasticity of bioflims can provide insight into the mechanics of biofilms Quantitative measure of the “strength” of a biofilm could be useful for: Development of drugs for inhibition of biofilm growth In identifying drug targets Characterizing the effect of specific molecular changes of biofilms.

21. Future work We want to understand fundamentally how theviscoelasticity changes on different lenght scales on differentfrequencies; We want to understand fundamentally how the viscoelasticity changes on different lenght scales on different frequencies; The methods will be first tested on water; The final testground will be viscoelastic characterization of bacterial biofilms at different stages of biofilm evolution. We will use optical tweezers to study viscoelastic properties of different biological samples;

22. References Annu. Rev. Biophys. Biomol. Struct. 1994. 23.’247-85 Annu. Rev. Condens. Matter Phys. 2010.1:301-322. Natan Osterman, Study of viscoelastic properties, interparticle potentials and self ordering in soft matter with magneto-optical tweezers, Doctoral thesis, University Ljubljana, 2009. Natan Osterman, TweezPal – Optical tweezers analysis and calibration software, Computer Physics Communications 181 (2010) 1911–1916 Oscar Björnham, A study of bacterial adhesion on a single – cell level by means of force measuring optical tweezers and simulations, Department of Applied Physics and Electronics, Umeå University, Sweden 2009 Mark C. Williams, Optical Tweezers: Measuring Piconewton Forces, Northeastern University