1. Fluorescence Fluctuation Spectroscopy – A tool for the detection of nanometer sized particles in living cells Michael Edetsberger Max F. Perutz Laboratories, Department for Biomolecular Structural Chemistry, University of Vienna

2. Introduction  Nanoparticles are extensively used in biotechnology and medicine  Nanoparticles originated from industrial or combustion processes  Nanoparticles play an important role in environmental biology, job safety and medicine

3. Standard techniques  Microscopy (Fluorescence, Laser Scanning)  Good spatial distribution  Diffraction limited  No information about dynamics and concentration  Electron Microscopy (TEM, EELS)  Good spatial distribution  Not diffraction limited  Cells have to be fixed and extensively stained  No information about dynamics and concentration

4. Outline  Fluorescence Fluctuation Spectroscopy (FFS)  Technical set up and physical models  Simulations to demonstrate the principle of FFS  Translocation of fluorescent 20nm particles  Laser Scanning Microscopy  Fluorescence Fluctuation Spectroscopy  Model for translocation

5. Confocal set up for Fluorescence Fluctuation Spectroscopy

7. Fluorescence Fluctuation Spectroscopy (FFS) Fluorescence Correlation Spectroscopy (FCS): length and number of bursts  shape of the Correlation curve Photon Counting Histogram (PCH): intensity and number of bursts  shape of the PCH curve

8. Principles Auto Correlation Function Photon Counting Histogram hydrodynamic size concentration specific brightness concentration

9. Individual diffusion – different size and equal brightness or mean intensity

10. Combined diffusion – different size and equal brightness mean intensity black and red together double concentrated dye (black) single concentrated

11. Individual diffusion – equal size and different brightness mean intensity or

12. Combined diffusion – equal size and different brightness mean intensity increases black and red together double concentrated carriers

13. Convolutions species 1 (1kHz and 270nM) species 2 (700kHz and 2.7nM) species 3 (1700kHz and 0.9 nM) convolutions measurements in the cytoplasm of a native HeLa cell

14. Information obtainable by Fluorescence Fluctuation Spectroscopy  Equally bright species can be differentiated by their diffusion time and their concentrations can be estimated by the amplitude-fraction of the Auto Correlation Function  Species of different brightness can be differentiated only by their diffusion times but no estimation of their concentration is possible  Only PCH gives the possibility to extract information about concentration and brightness

15. Results Incubation of HeLa cells with 20nm green fluorescent negatively charged polystyrene particles

16. Laser Scanning Images after 20 minutes incubation native HeLa cellGenistein treated HeLa cell

17. Fluorescence Correlation Spectroscopy (1)

18. Fluorescence Correlation Spectroscopy (2)

19. Fluorescence Intensity Distribution Analysis (1) 1 minute after adding the particles 5 minutes after adding the particles 15 minutes after adding the particles 30 minutes after adding the particles 60 minutes after adding the particles

20. Fluorescence Intensity Distribution Analysis (2)

21. Summary  Isolated particles are detected with a very short time-delay  Particles are detected in the cytoplasm or the nucleus whether the cell is native or not  Bigger and brighter particles are detected with a time-delay exclusively in the cytoplasm of native HeLa cells  Aggregates range from 200 to 600nm and suggest a 5-20% sphere packing

22. Model for translocation

23. Thank you