Transcript PPTX
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 • sensitivity limited by quenching and blinking Advantages of Absorption • can detect without labeling • wide choices of absorbing labels if labeling is needed • low effects from bleaching and blinking Why Not Using Absorption ? • small absorption cross-section at roomtemperature (~10-2 nm2 for common fluorophores) # of absorption = cross-section/diffraction limit that’s ~10-7 for laser at 300 nm • strong background Why Not Using Absorption ? • low background of fluorescence detection Why Not Using Absorption ? • strong background of absorption detection 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 TEMPORAL AVERAGING Lock-in Amplifier Raw Data FT Desired Signal Noise Lock-in Amplifier Raw Data Desired Signal Reference Lock-in Amplifier Raw Data Desired Signal integrate over a long time Reference Noises are cancelled due to orthogonality of sine functions. Modulating the Signal intensity • The noises are mostly with low frequencies. • Modulating the signal at high frequency to circumvent these noises. modulated signal noises frequency Ground State Depletion • Pump beam affects the absorption of the probe beam by depleting the ground state population. Pump Probe Experimental Setup • High frequency modulation to overcome the noises. Detection of a 20 nm Gold Nanoparticles δδP Detection of Atto647N Before bleaching After bleaching Photothermal Contrast Pump at the absorption band of the molecule. Photothermal Contrast Heat dissipation from the absorber Photothermal Contrast Change in local refractive index Photothermal Contrast Probe beam scattered due to locally inhomogeneous refractive index 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 Experimental Setup Modulate the heating beam for lock-in amplification 514 nm 800 nm Backward scattered geometry to reduce laser noise Simultaneous fluorescence measurement Photothermal Images of Gold Nanoparticles Photothermal Images of BHQ110T-BHQ1 BHQ1 Histogram of Survival Times Low bleaching rate! SPATIAL AVERAGING Experimental Setup for duel –color detection TDI Atto612Q Direction Background Subtraction before photobleaching after subtraction of photobleaching the two images Background Rejection by Polarization polarization parallel to molecule dipole polarization perpendiclar to molecule dipole Dual-Color Subtraction 633 nm abs 671 nm abs TDI 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. THANK YOU FOR YOUR ATTENTION. Potential for SM Quantitative Measurement