1. “Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins” M. Hoffmann, C. Eggeling,S. Jakobs, S.W. Hell JOURNAL CLUB PRESENTATION 2/13/2006 Mehmet Dogan

2. OUTLINE Background: – Resolution, –STED, –RESOLFT Photoswitching Characterization of switch kinetics of protein: asFP595 Demonstration of RESOLFT idea : ~100nm resolution Conclusions

3. Resolution Limit Abbe’s Diffraction Limit: Abbe’s Equation Modified for Fluorescence: Abbe Limit Saturation Factor

4. Requirements for Subdiffraction Resolution Large saturation factor –Either large I(x) –Or small saturation intensity I sat Spatial intensity zero I sat I(x) x saturated

5. Reversible Saturable Optical Fluorescent Transition (RESOLFT) A B At Equilibrium: Rate Equations: Normalized Populations:

6. Spatial Intensity Zero for Increased Resolution

7. A Subset : STED Stimulated Emission Depletion State A: Fluorescent State State B: Non-fluorescent ground state Stimulated Emission vs. Spontaneous Emission Too high saturation intensity  Photo induced damage

8. Alternative Approach: Reduced I sat Systems with weak spontaneous interstate conversions Remember: Photoswitchable Fluorophores: ssFP595 : Photochromic Fluorescent Protein ON State (A) : fluorescence-activated OFF State (B) : fluorescence-inhibited 450 nm 560 nm

9. Photoswitching Photoswitching of protein in E-coli with wide field epifluorescence microscope Photoswitching of thin protein layer on a 0.3 µm focal spot I y = 2 W/cm 2 I b =0.1 W/cm 2 I y = 4.4 W/cm 2 I b =3.6 W/cm 2 P y =3.3 nW P b =2.2 nW 8 orders of magnitude less than STED

10. Drawbacks 1)Low quantum yield: <1% 2)Incomplete OFF (15% fluorescence) 3)Photobleaching with cycling 4)Intensity to be adjusted for fluorescence settling

11. Effects of I y and I b on Inhibition I sat ~ 1 W/cm 2

12. Effect of I y Larger I y gives larger Residual Fluorescence Strong inhibition and small fluorescence settling time

13. Subdiffraction focal spots Solid lines: calculated Dashed lines: measured Focal spot with two offset peaks using phase plate y x

14. Effective PSF Calculated effective PSF using experimental values Calculated Effective PSF using theoretical values Incomplete inhibition of fluorescence at the periphery: 0.3

15. Imaging Test Samples Grooves on test slides with focused ion beam milling 10µm long 100nm wide 0.5-1µm deep Separation: 500nm Immersion into buffer with asFP595: Grooves filled by adsorption

16. scan a-c a-f d-e 20nm steps 50ms dwell time I y = 600W/cm 2 I b =30 W/cm 2

17. Conclusion Demonstration of resolution increase with photoswithing at low power New proteins should be engineered Challenges Low quantum yield (1%) Slow switching  requires ms integration Action cross-talk