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M. Hoffmann, C. Eggeling,S. Jakobs, S.W. Hell

“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. OUTLINE. Background: Resolution, STED, RESOLFT

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M. Hoffmann, C. Eggeling,S. Jakobs, S.W. Hell

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

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

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

  6. Spatial Intensity Zero for Increased Resolution

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

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

  9. Photoswitching Photoswitching of thin protein layer on a 0.3 µm focal spot Photoswitching of protein in E-coli with wide field epifluorescence microscope Py=3.3 nW Pb=2.2 nW Iy= 4.4 W/cm2 Ib=3.6 W/cm2 Iy= 2 W/cm2 Ib=0.1 W/cm2 8 orders of magnitude less than STED

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

  11. Effects of Iy and Ib on Inhibition Isat~ 1 W/cm2

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

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

  14. Effective PSF Calculated Effective PSF using theoretical values Calculated effective PSF using experimental 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 20nm steps 50ms dwell time Iy= 600W/cm2 Ib=30 W/cm2 a-c a-f d-e

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

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