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Single Molecule Absorption

Single Molecule Absorption. Speaker: Yu- Hsien Hwang Fu Date: 11/29. Single Molecule Detection. Fluorescence . http://en.wikipedia.org/wiki/Fluorophore. http://web.uvic.ca/ail/techniques/epi-fluorescence.html. Fluorescence Limitations. requiring fluorophore labeling

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Single Molecule Absorption

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  1. Single Molecule Absorption Speaker: Yu-Hsien Hwang Fu Date: 11/29

  2. Single Molecule Detection • Fluorescence http://en.wikipedia.org/wiki/Fluorophore http://web.uvic.ca/ail/techniques/epi-fluorescence.html

  3. Fluorescence Limitations • requiring fluorophore labeling • sensitivity limited by quenching and blinking

  4. Advantages of Absorption • can detect without labeling • wide choices of absorbing labels if labeling is needed • low effects from bleaching and blinking

  5. Why Not Using Absorption ? • small absorption cross-section at room-temperature (~10-2 nm2 for common fluorophores)# of absorption = cross-section/diffraction limitthat’s ~10-7 for laser at 300 nm • strong background

  6. Why Not Using Absorption ? • low background of fluorescence detection

  7. Why Not Using Absorption ? • strong background of absorption detection

  8. Averaging out the Noise • Temporally • Ground-State Depletion Microscopy Chong, S, Min, W, and Xie, XS (2011), Phys. Chem. Lett. 1, 3316 - 3322 • Photothermal Contrast Gaiduk, A, Yorulmaz, M, Rujgrok, PV, Orrit, M. (2010), Science. 330, 353 – 356 • Spatially • Optical Absorption Celebrant, M, Philipp, K, Renn, A, Sandoghdar, V. (2011), Nature Photonics. 5, 95 - 98

  9. Temporal Averaging

  10. Lock-in Amplifier Raw Data Desired Signal FT Noise

  11. Lock-in Amplifier Raw Data Desired Signal Reference

  12. Lock-in Amplifier Raw Data Desired Signal integrate over a long time Noises are cancelled due to orthogonality of sine functions. Reference

  13. Modulating the Signal • The noises are mostly with low frequencies. • Modulating the signal at high frequency to circumvent these noises. modulated signal intensity noises frequency

  14. Ground State Depletion • Pump beam affects the absorption of the probe beam by depleting the ground state population. Pump Probe

  15. Experimental Setup • High frequency modulation to overcome the noises.

  16. Detection of a 20 nm Gold Nanoparticles δδP

  17. Detection of Atto647N Before bleaching After bleaching

  18. Photothermal Contrast Pump at the absorption band of the molecule.

  19. Photothermal Contrast Heat dissipation from the absorber

  20. Photothermal Contrast Change in local refractive index

  21. Photothermal Contrast Probe beam scattered due to locally inhomogeneous refractive index

  22. Special Features for Photothermal Contrast • High probe power to suppress photon noises – the probe beam can be chosen out side of the molecule’s absorption band. • Glycerol as medium – low heat conductivity

  23. Experimental Setup Modulate the heating beam for lock-in amplification Backward scattered geometry to reduce laser noise 514 nm 800 nm Simultaneous fluorescence measurement

  24. Photothermal Images of Gold Nanoparticles

  25. Photothermal Images of BHQ1-10T-BHQ1 BHQ1 Histogram of Survival Times Low bleaching rate!

  26. Spatial Averaging

  27. Experimental Setup for duel –color detection TDI Atto612Q

  28. Direction Background Subtraction before photobleaching after photobleaching subtraction of the two images

  29. Background Rejection by Polarization polarization perpendiclar to molecule dipole polarization parallel to molecule dipole

  30. Dual-Color Subtraction 633 nm abs 671 nm abs TDI

  31. Conclusion • Background rejection is the main obstacle of single molecule absorption. • Lock-in amplification temporally averages out the noises. • Spatial averaging can be conducted with specious specified design.

  32. Thank you for your Attention.

  33. Potential for SM Quantitative Measurement

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